Methods and systems for treating cell proliferation disorders using plasmonics enhanced photospectral therapy (pepst) and exciton-plasmon enhanced phototherapy (epep)

ABSTRACT

The use of plasmonics enhanced photospectral therapy (PEPST) and exiton-plasmon enhanced phototherapy (EPEP) in the treatment of various cell proliferation disorders, and the PEPST and EPEP agents and probes used therein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. application Ser. No. 11/935,655,filed Nov. 6, 2007, and U.S. application Ser. No. 12/059,484, filed Mar.31, 2008; and Provisional Applications Ser. No. 61/042,561, filed Apr.4, 2008; 61/035,559, filed Mar. 11, 2008; and 61/080,140, filed Jul. 11,2008; 60/954,263, filed Aug. 6, 2007; and 61/030,437, filed Feb. 21,2008, the contents of each of which are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to methods and systems for treating cellproliferation disorders, that provide better distinction between normal,healthy cells and those cells suffering a cell proliferation disorder(hereafter “target cells”) and preferably that can be performed usingnon-invasive or minimally invasive techniques.

2. Discussion of the Background

Cell Proliferation Disorders

There are several types of cell proliferation disorders. Exemplary cellproliferation disorders may include, but are not limited to, cancer,bacterial infection, immune rejection response of organ transplant,solid tumors, viral infection, autoimmune disorders (such as arthritis,lupus, inflammatory bowel disease, Sjogrens syndrome, multiplesclerosis) or a combination thereof, as well as aplastic conditionswherein cell proliferation is low relative to healthy cells, such asaplastic anemia. Of these, cancer is perhaps the most well known. Theterm “cancer” generally refers to a diverse class of diseases that arecommonly characterized by an abnormal proliferation of the diseasedcells. A unifying thread in all known types of cancer is the acquisitionof abnormalities in the genetic material of the cancer cell and itsprogeny. Once a cell becomes cancerous, it will proliferate withoutrespect to normal limits, invading and destroying adjacent tissues, andmay even spread to distant anatomic sites through a process calledmetastasis. These life-threatening, malignant properties of cancersdifferentiate them from benign tumors, which are self-limited in theirgrowth and do not invade or metastasize.

The impact of cancer on society cannot be overstated. The disease mayaffect people at all ages, with a risk factor that significantlyincreases with a person's age. It has been one of the principal causesof death in developed countries and, as our population continues to age,it is expected to be an even greater threat to our society and economy.Therefore, finding cures and effective treatments for cancer has been,and remains, a priority within the biomedical research community.

Treatment Methods

There are two main types of reactions in phototherapy:

-   -   (1) Type I reactions involve electrons and hydrogen atoms, which        are transferred between photo-active molecules (also called        photosensitizers) and substrates or solvent molecules. Oxygen        may participate in subsequent reactions: e.g., psoralens in        photopheresis and PUVA.    -   (2) Type II reactions involve singlet oxygen formation by energy        transfer from PA molecules in the lowest triplet state to oxygen        in the ground state: e.g., photodynamic therapy (PDT)

Existing treatments for cell proliferation disorders such as cancerinclude surgery, chemotherapy, radiation therapy, immunotherapy,monoclonal antibody therapy, and several other lesser known methods. Thechoice of therapy usually depends on the location and severity of thedisorder, the stage of the disease, as well as the patient's response tothe treatment.

While some treatments may only seek to manage and alleviate symptoms ofthe disorder, the ultimate goal of any effective therapy is the completeremoval or cure of all disordered cells without damage to the rest ofthe body. With cancer, although surgery may sometimes accomplish thisgoal, the propensity of cancer cells to invade adjacent tissue or tospread to distant sites by microscopic metastasis often limits theeffectiveness of this option. Similarly, the effectiveness of currentchemotherapy is often limited by toxicity to other tissues in the body.Radiation therapy suffers from similar shortcomings as otheraforementioned treatment methods. Most of these cancer treatmentmethods, including radiation therapy, are known to cause damage to DNA,which if not repaired during a critical stage in mitosis, the splittingof the cell during cell proliferation, leads to a programmed cell death,i.e. apoptosis. Further, radiation tends to damage healthy cells, aswell as malignant tumor cells.

In one existing treatment known as extracorporeal photophoresis (ECP),excellent results have been observed since its initial approval by theFDA in 1988.

Extracorporeal photopheresis is a leukapheresis-based immunomodulatorytherapy that has been approved by the US Food and Drug Administrationfor the treatment of cutaneous T-cell lymphoma (CTCL). ECP, also knownas extracorporeal photochemotherapy, is performed at more than 150centers worldwide for multiple indications. Long-term follow-up data areavailable from many investigators that indicate ECP produces diseaseremission and improved survival for CTCL patients. In addition to CTCL,ECP has been shown to have efficacy in the treatment of other T-cellmediated disorders, including chronic graft versus host disease (GVHD)and solid organ transplant rejection. ECP use for the treatment ofautoimmune disease, such as systemic sclerosis and rheumatoid arthritis,is also being explored.

ECP is generally performed using the UVAR XTS Photopheresis Systemdeveloped by Therakos, Inc (Exton, Pa.). The process is performedthrough one intravenous access port and has 3 basic stages: (1)leukapheresis, (2) photoactivation, and (3) reinfusion, and takes 3-4hours to complete. A typical treatment session would resemble thefollowing sequence of events:

(1) One 16-gauge peripheral intravenous line or central venous access isestablished in the patient;

(2) Blood (225 mL) is passed through 3 cycles of leukapheresis, or 125mL of blood is passed through 6 cycles, depending on the patient'shematocrit value and body size. At the end of each leukapheresis cycle,the red blood cells and plasma are returned to the patient;

(3) The collected WBCs (including approximately 5% of the peripheralblood mononuclear cells) are mixed with heparin, saline, and8-methoxypsoralen (8-MOP), which intercalates into the DNA of thelymphocytes upon exposure to UVA light and makes them more susceptibleto apoptosis when exposed to UVA radiation;

(4) The mixture is passed as a 1-mm film through a sterile cassettesurrounded by UVA bulbs for 180 minutes, resulting in an average UVAexposure of 2 J/cm² per lymphocyte; and

(5) The treated WBC mixture is returned to the patient.

Over the past 20 years, on-going research has explored the mechanism ofaction of ECP. The combination of 8-MOP and UVA radiation causesapoptosis of the treated T cells and may cause preferential apoptosis ofactivated or abnormal T cells, thus targeting the pathogenic cells ofCTCL or GVHD. However, given that only a small percentage of the body'slymphocytes are treated, this seems unlikely to be the only mechanism ofaction.

Other evidence suggests that ECP also induces monocytes to differentiateinto dendritic cells capable of phagocytosing and processing theapoptotic T-cell antigens. When these activated dendritic cells arereinfused into the systemic circulation, they may cause a systemiccytotoxic CD8⁺ T-lymphocyte-mediated immune response to the processedapoptotic T-cell antigens.

Finally, animal studies indicate that photopheresis may induceantigen-specific regulatory T cells, which may lead to suppression ofallograft rejection or GVHD.

However, there are still many limitations to ECP. For example, ECPrequires patient to be connected to a machine for hours per treatment.It requires establishing peripheral intravenous line or central venousaccess, which may be difficult to do in certain disease states such assystemic sclerosis or arthritis. There is also a risk of infection atthe venous or central line site, or in the central line catheter.Further, it requires removing typically several hundred milliliters ofwhole blood from the patient, hence, the treatment is limited topatients who has sufficiently large initial volume of blood to bewithdrawn. The American Association of Blood Blanks recommend a limit ofextracorporeal volume to 15% of the patient's whole body blood volume.Therefore, the size of the volume that can be treated generally has tobe at least 40 kg or more. Risk of contracting blood-born pathogen(Hepatitis, HIV, etc.) due to exposure to contaminated operating systemis also a concern.

Alternatively, a patient can be treated in vivo with a photosensitiveagent followed by the withdrawal of a sample from the patient, treatmentwith UV radiation in vitro (ex vivo), and reinjecting the patient withthe treated sample. This method is known for producing an autovaccine. Amethod of treating a patient with a photosensitive agent, exposing thepatient to an energy source and generating an autovaccine effect whereinall steps are conducted in vivo has not been described. See WO03/049801, U.S. Pat. No. 6,569,467; U.S. Pat. No. 6,204,058; U.S. Pat.No. 5,980,954; U.S. Pat. No. 6,669,965; U.S. Pat. No. 4,838,852; U.S.Pat. No. 7,045,124, and U.S. Pat. No. 6,849,058. Moreover, the sideeffects of extracorporeal photopheresis are well known and includenausea, vomiting, cutaneous erythema, hypersensitivity to sunlight, andsecondary hematologic malignancy. Researchers are attempting to usephotopheresis in experimental treatments for patients with cardiac,pulmonary and renal allograft rejection; autoimmune diseases, andulcerative colitis.

A survey of known treatment methods reveals that these methods tend toface a primary difficulty of differentiating between normal cells andtarget cells when delivering treatment, often due to the production ofsinglet oxygen which is known to be non-selective in its attack ofcells, as well as the need to perform the processes ex vivo, or throughhighly invasive procedures, such as surgical procedures in order toreach tissues more than a few centimeters deep within the subject.

U.S. Pat. No. 5,829,448 describes simultaneous two-photon excitation ofphoto-agents using irradiation with low-energy photons such as infraredor near infrared light (NRI). A single photon and simultaneoustwo-photon excitation is compared for psoralen derivatives, whereincells are treated with the photo agent and are irradiated with NRI or UVradiation. The patent suggests that treating with a low energyirradiation is advantageous because it is absorbed and scattered to alesser extent than UV radiation. However, the use of NRI or UV radiationis known to penetrate tissue to only a depth of a few centimeters. Thusany treatment deep within the subject would necessarily require the useof ex vivo methods or highly invasive techniques to allow theirradiation source to reach the tissue of interest.

Chen et al., J. Nanosci. and Nanotech., 6:1159-1166 (2006); Kim et al.,JACS, 129:2669-2675 (2007); U.S. 2002/0127224; and U.S. Pat. No.4,979,935 each describe methods for treatment using various types ofenergy activation of agents within a subject. However, each suffers fromthe drawback that the treatment is dependent on the production ofsinglet oxygen to produce the desired effect on the tissue beingtreated, and is thus largely indiscriminate in affecting both healthycells and the diseased tissue desired to be treated.

U.S. Pat. No. 6,908,591 discloses methods for sterilizing tissue withirradiation to reduce the level of one or more active biologicalcontaminants or pathogens, such as viruses, bacteria, yeasts, molds,fungi, spores, prions or similar agents responsible, alone or incombination, for transmissible spongiform encephalopathies and/or singleor multicellular parasites, such that the tissue may subsequently beused in transplantation to replace diseased and/or otherwise defectivetissue in an animal. The method may include the use of a sensitizer suchas psoralen, a psoralen-derivative or other photosensitizer in order toimprove the effectiveness of the irradiation or to reduce the exposurenecessary to sterilize the tissue. However, the method is not suitablefor treating a patient and does not teach any mechanisms for stimulatingthe photosensitizers, indirectly.

U.S. Pat. No. 6,235,508 discloses antiviral applications for psoralensand other photoactivatable molecules. It teaches a method forinactivating viral and bacterial contaminants from a biologicalsolution. The method includes mixing blood with a photosensitizer and ablocking agent and irradiating the mixture to stimulate thephotosensitizer, inactivating substantially all of the contaminants inthe blood, without destroying the red blood cells. The blocking agentprevents or reduces deleterious side reactions of the photosensitizer,which would occur if not in the presence of the blocking agent. The modeof action of the blocking agent is not predominantly in the quenching ofany reactive oxygen species, according to the reference.

Also, U.S. Pat. No. 6,235,508 suggests that halogenated photosensitizersand blocking agents might be suitable for replacing 8-methoxypsoralen(8-MOP) in photophoresis and in treatment of certain proliferativecancers, especially solid localized tumors accessible via a fiber opticlight device or superficial skin cancers. However, the reference failsto address any specific molecules for use in treating lymphomas or anyother cancer. Instead, the reference suggests a process of photophoresisfor antiviral treatments of raw blood and plasma.

U.S. Pat. No. 6,235,508 teaches away from 8-MOP and4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) and many otherphotoactivatable molecules, which are taught to have certaindisadvantages. Fluorescing photosensitizers are said to be preferred,but the reference does not teach how to select a system of fluorescentstimulation or photoactivation using fluorescent photosensitizers.Instead, the fluorescing photosensitizer is limited to the intercalatorthat is binding to the DNA. The reference suggests that fluorescenceindicates that such an intercalator is less likely to stimulate oxygenradicals. Thus, the reference fails to disclose any mechanism ofphotoactivation of an intercalator other than by direct photoactivationby UV light, although use of a UV light probe or X-rays is suggested forpenetrating deeper into tissues. No examples are provided for the use ofa UV light probe or for use of X-rays. No example of any stimulation byX-ray radiation is taught.

Psoralens and Related Compounds

U.S. Pat. No. 6,235,508 further teaches that psoralens are naturallyoccurring compounds which have been used therapeutically for millenniain Asia and Africa. The action of psoralens and light has been used totreat vitiligo and psoriasis (PUVA therapy; Psoralen Ultra Violet A).Psoralen is capable of binding to nucleic acid double helices byintercalation between base pairs; adenine, guanine, cytosine and thymine(DNA) or uracil (RNA). Upon sequential absorption of two UV-A photons,psoralen in its excited state reacts with a thymine or uracil doublebond and covalently attaches to both strands of a nucleic acid helix.The crosslinking reaction appears to be specific for a thymine (DNA) ora uracil (RNA) base. Binding proceeds only if psoralen is intercalatedin a site containing thymine or uracil, but an initial photoadduct mustabsorb a second UVA photon to react with a second thymine or uracil onthe opposing strand of the double helix in order to crosslink each ofthe two strands of the double helix, as shown below. This is asequential absorption of two single photons as shown, as opposed tosimultaneous absorption of two or more photons.

In addition, the reference teaches that 8-MOP is unsuitable for use asan antiviral, because it damages both cells and viruses. Lethal damageto a cell or virus occurs when the psoralen is intercalated into anucleic acid duplex in sites containing two thymines (or uracils) onopposing strands but only when it sequentially absorbs 2 UVA photons andthymines (or uracils) are present. U.S. Pat. No. 4,748,120 of Wiesehanis an example of the use of certain substituted psoralens by aphotochemical decontamination process for the treatment of blood orblood products.

Additives, such as antioxidants are sometimes used with psoralens, suchas 8-MOP, AMT and I-IMT, to scavenge singlet oxygen and other highlyreactive oxygen species formed during photoactivation of the psoralens.It is well known that UV activation creates such reactive oxygenspecies, which are capable of seriously damaging otherwise healthycells. Much of the viral deactivation may be the result of thesereactive oxygen species rather than any effect of photoactivation ofpsoralens. Regardless, it is believed that no auto vaccine effect hasbeen observed.

The best known photoactivatable compounds are derivatives of psoralen orcoumarin, which are nucleic acid intercalators. The use of psoralen andcoumarin photosensitizers can give rise to alternative chemical pathwaysfor dissipation of the excited state that are either not beneficial tothe goal of viral inactivation, or that are actually detrimental to theprocess. For psoralens and coumarins, this chemical pathway is likely tolead to the formation of a variety of ring-opened species, such as shownbelow for coumarin:

Research in this field over-simplifies mechanisms involved in thephotoactivating mechanism and formation of highly reactive oxygenspecies, such as singlet oxygen. Both may lead to inactivating damage oftumor cells, viruses and healthy cells. However, neither, alone orcombined, lead to an auto vaccine effect. This requires an activation ofthe body's own immune system to identify a malignant cell or virus asthreat and to create an immune response capable of lasting cytotoxiceffects directed to that threat. It is believed, without being limitingin any way, that photoactivation and the resulting apoptosis ofmalignant cells that occurs in extracorporeal photophoresis causes theactivation of an immune response with cytotoxic effects on untreatedmalignant cells. While the complexity of the immune response andcytotoxid effects is fully appreciated by researchers, a therapy thatharnesses the system to successfully stimulate an auto vaccine effectagainst a targeted, malignant cell has been elusive, except forextracorporeal photophoresis for treating lymphoma.

Midden (W. R. Midden, Psoralen DNA photobiology, Vol I1 (ed. F. P.Gaspalloco) CRC press, pp. 1. (1988) has presented evidence thatpsoralens photoreact with unsaturated lipids and photoreact withmolecular oxygen to produce active oxygen species such as superoxide andsinglet oxygen that cause lethal damage to membranes. U.S. Pat. No.6,235,508 teaches that 8-MOP and AMT are unacceptable photosensitizers,because each indiscriminately damages both cells and viruses. Studies ofthe effects of cationic side chains on furocoumarins as photosensitizersare reviewed in Psoralen DNA Photobiology, Vol. I, ed. F. Gaspano, CRCPress, Inc., Boca Raton, Fla., Chapter 2. U.S. Pat. No. 6,235,508 gleansthe following from this review: most of the amino compounds had a muchlower ability to both bind and form crosslinks to DNA compared to 8-MOP,suggesting that the primary amino functionality is the preferred ionicspecies for both photobinding and crosslinking.

U.S. Pat. No. 5,216,176 of Heindel discloses a large number of psoralensand coumarins that have some effectiveness as photoactivated inhibitorsof epidermal growth factor. Halogens and amines are included among thevast functionalities that could be included in the psoralen/coumarinbackbone. This reference is incorporated herein by reference.

U.S. Pat. No. 5,984,887 discloses using extracorporeal photophoresiswith 8-MOP to treat blood infected with CMV. The treated cells as wellas killed and/or attenuated virus, peptides, native subunits of thevirus itself (which are released upon cell break-up and/or shed into theblood) and/or pathogenic noninfectious viruses are then used to generatean immune response against the virus, which was not present prior to thetreatment.

Photodynamic Therapy (PDT)

PDT is a relatively new light-based treatment, which has recently beenapproved by the United States Food & Drug Administration (FDA) for thetreatment of both early and late-stage lung cancer. Other countries haveapproved PDT for treatment of various cancers as well. Unlikechemotherapy, radiation, and surgery, PDT is useful in treating all celltypes, whether small cell or non-small cell carcinoma. PDT involvestreatment of diseases such as cancer using light action on a specialphotoactive class of drugs, by photodynamic action in vivo to destroy ormodify tissue [Dougherty T. J. and Levy J. G., “Photodynamic Therapy andClinical Applications”, in Biomedical Photonics Handbook, Vo-Dinh T.,Ed., CRC Press, Boca Raton Fla. (2003)]. PDT, which was originallydeveloped for treatment of various cancers, has now been used to includetreatment of pre-cancerous conditions, e.g. actinic keratoses,high-grade dysplasia in Barrett's esophagus, and non-cancerousconditions, e.g. various eye diseases, e.g. age related maculardegeneration (AMD). Photodynamic therapy (PDT) is approved forcommercialization worldwide both for various cancers (lung, esophagus)and for AMD.

The PDT process requires three elements: (1) a PA drug (i.e.,photosensitizer), (2) light that can excite the photosensitizer and (3)endogenous oxygen. The putative cytotoxic agent is singlet oxygen, anelectronically excited state of ground state triplet oxygen formedaccording to the Type II photochemical process, as follows.

PA+hν→¹PA*(S) Excitation¹PA*(S)→³PA*(T) Intersystem crossing for singlet to triplet state³PA*(T)→O₂→¹O*₂+PA Energy transfer from the drug to singlet oxygenwhere PA=photo-active drug at the ground state; ¹PA*(S)=excited singletstate; ³PA*(T)=excited triplet state; ¹O*₂=singlet excited state ofoxygen

Because the triplet state has a relatively long lifetime (μsec toseconds) only photosensitizers that undergo efficient intersystemcrossing to the excited triplet state will have sufficient time forcollision with oxygen in order to produce singlet oxygen. The energydifference between ground state and singlet oxygen is 94.2 kJ/mol andcorresponds to a transition in the near-infrared at ˜1270 nm. Most PAphotosensitizers in clinical use have triplet quantum yields in therange of 40-60% with the singlet oxygen yield being slightly lower.Competing processes include loss of energy by deactivation to groundstate by fluorescence or internal conversion (loss of energy to theenvironment or surrounding medium).

However, while a high yield of singlet oxygen is desirable it is by nomeans sufficient for a photosensitizer to be clinically useful.Pharmacokinetics, pharmacodynamics, stability in vivo and acceptabletoxicity play critical roles as well [Henderson B W, Gollnick S O,“Mechanistic Principles of Photodynamic Therapy”, in BiomedicalPhotonics Handbook, Vo-Dinh T, Ed., CRC Press, Boca Raton Fla. (2003)].For example, it is desirable to have relatively selective uptake in thetumor or other tissue being treated relative to the normal tissue thatnecessarily will be exposed to the exciting light as well.Pharmacodynamic issues such as the subcellular localization of thephotosensitizer may be important as certain organelles appear to be moresensitive to PDT damage than others (e.g. the mitochondria). Toxicitycan become an issue if high doses of photosensitizer are necessary inorder to obtain a complete response to treatment. An important mechanismassociated with PDT drug activity involves apoptosis in cells. Uponabsorption of light, the photosensitiser (PS) initiates chemicalreactions that lead to the direct or indirect production of cytotoxicspecies such as radicals and singlet oxygen. The reaction of thecytotoxic species with subcellular organelles and macromolecules(proteins, DNA, etc) lead to apoptosis and/or necrosis of the cellshosting the PDT drug. The preferential accumulation of PDT drugmolecules in cancer cells combined with the localized delivery of lightto the tumor, results in the selective destruction of the cancerouslesion. Compared to other traditional anticancer therapies, PDT does notinvolve generalized destruction of healthy cells. In addition to directcell killing, PDT can also act on the vasculature, reducing blood flowto the tumor causing its necrosis. In particular cases it can be used asa less invasive alternative to surgery.

There are several chemical species used for PDT includingporphyrin-based sensitizers. A purified hematoporphyrin derivative,Photofrin®, has received approval of the US Food and DrugAdministration. Porphyrins are generally used for tumors on or justunder the skin or on the lining of internal organs or cavities becausetheses drug molecules absorbs light shorter than 640 nm in wavelength.For tumors occurring deep in tissue, second generation sensitizers,which have absorbance in the NIR region, such as porphyrin-based systems[R. K. Pandey, “Synthetic Strategies in designing Porphyrin-BasedPhotosensitizers', in Biomedical Photonics Handbook, Vo-Dinh T., Ed.,CRC Press, Boca Raton Fla. (2003)], chlorines, phthalocyanine, andnaphthalocyanine have been investigated.

PDT retains several photosensitizers in tumors for a longer time than innormal tissues, thus offering potential improvement in treatmentselectivity. See Corner C., “Determination of [3H]- and [14C]hematoporphyrin derivative distribution in malignant and normal tissue,”Cancer Res 1979, 3 9: 146-15 1; Young S W, et al., “Lutetium texaphyrin(PCI-0123) a near-infrared, water-soluble photosensitizer,” PhotochemPhotobiol 1996, 63:892-897; and Berenbaum M C, et al.,“Meso-Tetra(hydroxyphenyl)porphyrins, a new class of potent tumorphotosensitisers with favourable selectivity,” Br J Cancer 1986,54:717-725. Photodynamic therapy uses light of a specific wavelength toactivate the photosensitizing agent. Various light sources have beendeveloped for PDT, which include dye lasers and diode lasers. Lightgenerated by lasers can be coupled to optical fibers that allow thelight to be transmitted to the desired site. See Pass 1-11,“Photodynamic therapy in oncology: mechanisms and clinical use,” J NatlCancer Inst 1993, 85:443-456. According to researchers, the cytotoxiceffect of PDT is the result of photooxidation reactions, as disclosed inFoote C S, “Mechanisms of photooxygenation,” Proa Clin Biol Res 1984,170:3-18. Light causes excitation of the photosensitizer, in thepresence of oxygen, to produce various toxic species, such as singletoxygen and hydroxyl radicals. It is not clear that direct damage to DNAis a major effect; therefore, this may indicate that photoactivation ofDNA crosslinking is not stimulated efficiently.

Furthermore, when laser light is administered via external illuminationof tissue surfaces, the treatment effect of PDT is confined to a fewmillimeters (i.e. superficial). The reason for this superficiallimitation is mainly the limited penetration of the visible light usedto activate the photosensitizer. Thus, PDT is used to treat the surfacesof critical organs, such as lungs or intra-abdominal organs, withoutdamage to the underlying structures. However, even these treatmentsrequire significantly invasive techniques to treat the surface of theaffected organs. Clinical situations use the procedure in conjunctionwith surgical debulking to destroy remnants of microscopic or minimalgross disease. It is possible that the laser light and small amount ofremaining microscopic and minimal gross disease results in too little orhighly damaged structures. Pre-clinical data show that some immuneresponse is generated, but clinical trials have reported no auto vaccineeffect similar to that produced by extracorporeal photophoresis inclinical conditions. Instead, the immune response appears to be vigorousonly under limited conditions and only for a limited duration.

Problems

It is well recognized that a major problem associated with the existingmethods of diagnosis and treatment of cell proliferation disorders is indifferentiation of normal cells from target cells. Such targetspecificity is difficult to achieve by way of surgery since the strategythere is simply to cut out a large enough portion of the affected areato include all diseased cells and hope that no diseased cells havespread to other distant locations.

With chemotherapy, while some degree of differentiation can be achieved,healthy cells are generally adversely affected by chemo-agents. As insurgery, the treatment strategy in chemotherapy is also to kill off alarge population of cells, with the understanding that there are farmore normal cells than diseased cells so that the organism can recoverfrom the chemical assault.

Radiation therapy works by irradiating cells with high levels of highenergy radiation such as high-energy photon, electron, or proton. Thesehigh energy beams ionize the atoms which make up a DNA chain, which inturn leads to cell death. Unlike surgery, radiation therapy does notrequire placing patients under anesthesia and has the ability to treattumors deep inside the body with minimal invasion of the body. However,the high doses of radiation needed for such therapies damages healthycells just as effectively as it does diseased cells. Thus, similar tosurgery, differentiation between healthy and diseased cells in radiationtherapy is only by way of location. There is no intrinsic means for aradiation beam to differentiate between a healthy cell from a diseasedcell either.

Other methods may be more refined. For example, one form of advancedtreatment for lymphoma known as extracorporeal photopheresis involvesdrawing the patient's blood from his body into an instrument where thewhite cells (buffy coat) are separated from the plasma and the red bloodcells. A small amount of the plasma separated in this process is thenisolated and mixed with a photosensitizer (PS), a drug that can beactivated by light. The buffy coat is then exposed to a light toactivate the drug. The treated blood is then returned to the patient. Inthis example, one may think of the target-specificity problem as beingsolved by separating the blood from the rest of the body where thetarget components are easily exposed.

However, this procedure has its drawbacks; it requires drawing bloodfrom the patient, thus requiring cumbersome machinery to perform and mayrequire blood transfusion in order to maintain the volume of blood flowin the machine. Further, this also limits the size of the patient thatcan be treated, since the extracorporeal volume is great and too muchwithdrawal of blood increases the risk of hypovolemic shock. The methodis also limited to treating blood-born cell proliferation relateddisorders such as lymphoma, and is not capable of treating solid tumorsor other types of non-blood related cell proliferation disorders.

A problem encountered in PDT therapy is the inability to treat targetareas that are more than a few centimeters beneath the surface of theskin without significant invasive techniques, and the fact that PDTtypically operates by generation of sufficient quantities of singletoxygen to cause cell lysis. However, singlet oxygen in sufficientconcentration will lyse not only target cells, but also healthy cellsrather indiscriminately.

Therefore, there still exists a need for better and more effectivetreatments that can more precisely target the diseased cells withoutcausing substantial side-effects or collateral damages to healthytissues, and which are capable of treating even solid tumors or othertypes of non-blood related cell proliferation disorders.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodfor the treatment of a cell proliferation disorder that permitstreatment of a subject in any area of the body while being non-invasiveand having high selectivity for targeted cells relative to healthy cellsthrough the use of plasmonics materials.

A further object of the present invention is to provide a method fortreatment of a cell proliferation disorder which can use any suitableenergy source as the initiation energy source in combination withplasmonics materials to activate the activatable pharmaceutical agentand thereby cause a predetermined cellular change to treat cellssuffering from a cell proliferation disorder.

A further object of the present invention is to provide a method fortreatment of a cell proliferation disorder using plasmonics in an energycascade to activate an activatable pharmaceutical agent that then treatscells suffering from a cell proliferation disorder.

A further object of the present invention is to provide a method fortreatment of a cell proliferation disorder using an energy cascade thathas amplified electromagnetic fields to activate an activatablepharmaceutical agent that then treats cells suffering from a cellproliferation disorder.

A further object of the present invention is to provide a method for thetreatment of a cell proliferation disorder that permits treatment of asubject in any area of the body while being non-invasive and having highselectivity for targeted cells relative to healthy cells through the useof exciton-plasmon enhancement.

A further object of the present invention is to provide a method fortreatment of a cell proliferation disorder which can use any suitableenergy source as the initiation energy source in combination withexciton-plasmon enhancement to activate the activatable pharmaceuticalagent and thereby cause a predetermined cellular change to treat cellssuffering from a cell proliferation disorder.

A further object of the present invention is to provide a method fortreatment of a cell proliferation disorder using exciton-plasmonenhancement in an energy cascade to activate an activatablepharmaceutical agent that then treats cells suffering from a cellproliferation disorder.

These and other objects of the present invention, which will become moreapparent in conjunction with the following detailed description of thepreferred embodiments, either alone or in combinations thereof, havebeen satisfied by the discovery of a method for treating a cellproliferation disorder in a subject, comprising:

-   -   (1) administering to the subject at least one activatable        pharmaceutical agent that is capable of effecting a        predetermined cellular change when activated and at least one        plasmonics agent; and    -   (2) applying an initiation energy from an initiation energy        source to the subject, wherein the plasmonics agent enhances the        applied initiation energy, such that the enhanced initiation        energy activates the activatable agent in situ,    -   thus causing the predetermined cellular change to occur, wherein        said predetermined cellular change treats the cell proliferation        related disorder.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a graphical representation of plasmonic nanostructures andtheir theoretical electromagnetic enhancement at different excitationwavelengths.

FIG. 2 provides representative embodiments of plasmonics photo-activeprobes useful in the present invention.

FIG. 3 is a graphical explanation of the plasmonics-enhanced effect ofphotospectral therapy used in the present invention.

FIG. 4 provides representative embodiments of plasmonics-activenanostructures.

FIG. 5 is a graphical representation of one embodiment of a PEPST probewith remote drug release.

FIG. 6 is a graphical representation of several embodiments of PEPSTprobes with various linkers for remote drug release.

FIG. 7 is a graphical representation of several embodiments ofplasmonics photo-active probes with bioreceptors.

FIG. 8 is a graphical representation of the “therapeutic window” intissue and absorption spectra of biological components.

FIG. 9 is a graphical representation of an embodiment of the energymodulation agent (or excitation energy converter/EEC)-photo activator(PA) system of the present invention.

FIG. 10 is a graphical representation of several embodiments ofplasmonics photo-active energy modulation agent-PA probes.

FIG. 11 shows structures of various preferred embodiments of goldcomplexes exhibiting XEOL.

FIG. 12 shows the structure of a further embodiment of compoundexhibiting XEOL, namely a tris-8-hydroxyquinoline-aluminum complex.

FIG. 13 is a graphical representation of a plasmonics-enhanced mechanismfor a photo-active energy modulation agent-PA probe of the presentinvention.

FIG. 14 is a graph showing excitation and emission fluorescence spectraof psoralens.

FIG. 15 is a graphical representation of an embodiment of a PEPST energymodulation agent-PA system with detachable bond.

FIG. 16 is a graphical representation of an embodiment of PEPST probesfor dual plasmonic excitation.

FIG. 17 is a graphical representation of an embodiment of a use ofencapsulated photoactive agents.

FIG. 18 is a simplified graphical representation of the use of thepresent invention principle of non-invasive PEPST modality.

FIG. 19 is an photomicrograph showing nanocaps (half-nanoshells)comprising polystyrene nanospheres coated with silver.

FIG. 20 shows various schematic embodiments of basic EIP probes.

FIG. 21 is a graphical representation of fluorescence spectra of PAHcompounds.

FIG. 22 is a graph showing the XEOL of Eu doped in BaFBr matrix.

FIG. 23 provides further embodiments of schematic designs of EIP probes.

FIG. 24 is a graphical representation of various embodiments of basicEPEP probes.

FIG. 25 is a graphical representation of various embodiments of EPEPprobes having NPs, NWs and NRs.

FIG. 26 is a graphical representation of various embodiments of EPEPprobes having NPs, NWs, NRs and bioreceptors.

FIG. 27 is a graphical representation of an embodiment of EPEP probeshaving NPs and multiple NWs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention sets forth a novel method of treating cellproliferation disorders that is effective, specific, and has fewside-effects. Those cells suffering from a cell proliferation disorderare referred to herein as the target cells. A treatment for cellproliferation disorders, including solid tumors, is capable ofchemically binding cellular nucleic acids, including but not limited to,the DNA or mitochondrial DNA or RNA of the target cells. For example, aphotoactivatable agent, such as a psoralen or a psoralen derivative, isexposed in situ to an energy source capable of activating thephotoactivatable agent or agents selected. In another example, thephotoactivatable agent is a photosensitizer. The photoactivatable agentmay be a metal nanocluster or a molecule or group of molecules, or acombination thereof. In particular, the present invention method takesadvantage of the unique properties of plasmonics to enhance thephotospectral therapy methods described in U.S. Ser. No. 11/935,655,filed Nov. 6, 2007 by one of the current inventors, the entire contentsof which are hereby incorporated by reference. A preferred embodiment ofthe present invention is thus called “plasmonics-enhanced photospectraltherapy” or PEPST for short.

As noted above, an object of the present invention is to treat cellproliferation disorders. Exemplary cell proliferation disorders mayinclude, but are not limited to, cancer, as well as bacterial and viralinfections where the invading bacteria grows at a much more rapid ratethan cells of the infected host. In addition, treatment for certaindevelopmental stage diseases related to cell proliferation, such assyndactyl), are also contemplated. Accordingly, in one embodiment, thepresent invention provides methods that are capable of overcoming theshortcomings of the existing methods. In general, a method in accordancewith the present invention utilizes the principle of energy transfer orenergy excitation, to and among molecular agents to control delivery andactivation of pharmaceutically active agents such that delivery of thedesired pharmacological effect is more focused, precise, and effectivethan the conventional techniques.

Generally, the present invention provides methods for the treatment ofcell proliferation disorders, in which an initiation energy sourceprovides an initiation energy that activates an activatablepharmaceutical agent to treat target cells within the subject. In onepreferred embodiment, the initiation energy source is applied indirectlyto the activatable pharmaceutical agent, preferably in proximity to thetarget cells. Within the context of the present invention, the phrase“applied indirectly” (or variants of this phrase, such as “applyingindirectly”, “indirectly applies”, “indirectly applied”, “indirectlyapplying”, etc.), when referring to the application of the initiationenergy, means the penetration by the initiation energy into the subjectbeneath the surface of the subject and to the activatable pharmaceuticalagent within a subject.

Although not intending to be bound by any particular theory or beotherwise limited in any way, the following theoretical discussion ofscientific principles and definitions are provided to help the readergain an understanding and appreciation of the present invention. As usedherein, the term “subject” is not intended to be limited to humans, butmay also include animals, plants, or any suitable biological organism.

As used herein, the phrase “cell proliferation disorder” refers to anycondition where the growth rate of a population of cells is less than orgreater than a desired rate under a given physiological state andconditions. Although, preferably, the proliferation rate that would beof interest for treatment purposes is faster than a desired rate, slowerthan desired rate conditions may also be treated by methods of thepresent invention. Exemplary cell proliferation disorders may include,but are not limited to, cancer, bacterial infection, immune rejectionresponse of organ transplant, solid tumors, viral infection, autoimmunedisorders (such as arthritis, lupus, inflammatory bowel disease,Sjogrens syndrome, multiple sclerosis) or a combination thereof, as wellas aplastic conditions wherein cell proliferation is low relative tohealthy cells, such as aplastic anemia. Particularly preferred cellproliferation disorders for treatment using the present methods arecancer, staphylococcus aureus (particularly antibiotic resistant strainssuch as methicillin resistant staphylococcus aureus or MRSA), andautoimmune disorders.

As used herein, an “activatable pharmaceutical agent” (alternativelycalled a “photoactive agent” or PA) is an agent that normally exists inan inactive state in the absence of an activation signal. When the agentis activated by a matching activation signal under activatingconditions, it is capable of effecting the desired pharmacologicaleffect on a target cell (i.e. preferably a predetermined cellularchange).

Signals that may be used to activate a corresponding agent may include,but are not limited to, photons of specific wavelengths (e.g. x-rays, orvisible light), electromagnetic energy (e.g. radio or microwave),thermal energy, acoustic energy, or any combination thereof.

Activation of the agent may be as simple as delivering the signal to theagent or may further premise on a set of activation conditions. Forexample, in the former case, an activatable pharmaceutical agent, suchas a photosensitizer, may be activated by UV-A radiation. Onceactivated, the agent in its active-state may then directly proceed toeffect a cellular change.

Where activation may further premise upon other conditions, meredelivery of the activation signal may not be sufficient to bring aboutthe desired cellular change. For example, a photoactive compound thatachieves its pharmaceutical effect by binding to certain cellularstructure in its active state may require physical proximity to thetarget cellular structure when the activation signal is delivered. Forsuch activatable agents, delivery of the activation signal undernon-activating conditions will not result in the desired pharmacologiceffect. Some examples of activating conditions may include, but are notlimited to, temperature, pH, location, state of the cell, presence orabsence of co-factors. Selection of an activatable pharmaceutical agentgreatly depends on a number of factors such as the desired cellularchange, the desired form of activation, as well as the physical andbiochemical constraints that may apply. Exemplary activatablepharmaceutical agents may include, but are not limited to, agents thatmay be activated by photonic (electromagnetic) energy, acoustic energy,chemical or enzymatic reactions, thermal energy, or any other suitableactivation mechanisms.

When activated, the activatable pharmaceutical agent may effect cellularchanges that include, but are not limited to, apoptosis, redirection ofmetabolic pathways, up-regulation of certain genes, down-regulation ofcertain genes, secretion of cytokines, alteration of cytokine receptorresponses, production of reactive oxygen species or combinationsthereof.

The mechanisms by which an activatable pharmaceutical agent may achieveits desired effect are not particularly limited. Such mechanisms mayinclude direct action on a predetermined target as well as indirectactions via alterations to the biochemical pathways. A preferred directaction mechanism is by binding the agent to a critical cellularstructure such as nuclear DNA, mRNA, rRNA, ribosome, mitochondrial DNA,or any other functionally important structures. Indirect mechanisms mayinclude releasing metabolites upon activation to interfere with normalmetabolic pathways, releasing chemical signals (e.g. agonists orantagonists) upon activation to alter the targeted cellular response,and other suitable biochemical or metabolic alterations.

The treatment of the present invention can be by the unique methodsdescribed in U.S. application Ser. No. 11/935,655, filed Nov. 6, 2007(incorporated by reference above), or by a modified version of aconventional treatment such as PDT, but using a plasmonics-active agentto enhance the treatment by modifying or enhancing the applied energyor, in the case of using an energy modulation agent, modifying eitherthe applied energy, the emitted energy from the energy modulation agent,or both.

In one preferred embodiment, the activatable pharmaceutical agent iscapable of chemically binding to the DNA or mitochondria at atherapeutically effective amount. In this embodiment, the activatablepharmaceutical agent, preferably a photoactivatable agent, is exposed insitu to an activating energy emitted from an energy modulation agent,which, in turn receives energy from an initiation energy source.

Suitable activatable agents include, but are not limited to, photoactiveagents, sono-active agents, thermo-active agents, andradio/microwave-active agents. An activatable agent may be a smallmolecule; a biological molecule such as a protein, a nucleic acid orlipid; a supramolecular assembly; a nanoparticle; a nanostructure, orcombinations thereof; or any other molecular entity having apharmaceutical activity once activated.

The activatable agent may be derived from a natural or synthetic origin.Any such molecular entity that may be activated by a suitable activationsignal source to effect a predetermined cellular change may beadvantageously employed in the present invention. Suitable photoactiveagents include, but are not limited to: psoralens and psoralenderivatives, pyrene cholesteryloleate, acridine, porphyrin, fluorescein,rhodamine, 16-diazorcortisone, ethidium, transition metal complexes ofbleomycin, transition metal complexes of deglycobleomycin,organoplatinum complexes, alloxazines such as 7,8-dimethyl-10-ribitylisoalloxazine (riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin),7,8-dimethylalloxazine (lumichrome), isoalloxazine-adenine dinucleotide(flavine adenine dinucleotide [FAD]), alloxazine mononucleotide (alsoknown as flavine mononucleotide [FMN] and riboflavine-5-phosphate),vitamin Ks, vitamin L, their metabolites and precursors, andnapththoquinones, naphthalenes, naphthols and their derivatives havingplanar molecular conformations, porphyrins, dyes such as neutral red,methylene blue, acridine, toluidines, flavine (acriflavinehydrochloride) and phenothiazine derivatives, coumarins, quinolones,quinones, and anthroquinones, aluminum (111) phthalocyaninetetrasulfonate, hematoporphyrin, and phthalocyanine, and compounds whichpreferentially adsorb to nucleic acids with little or no effect onproteins. The term “alloxazine” includes isoalloxazines.

Endogenously-based derivatives include synthetically derived analogs andhomologs of endogenous photoactivated molecules, which may have or lacklower (1 to 5 carbons) alkyl or halogen substituents of thephotosensitizers from which they are derived, and which preserve thefunction and substantial non-toxicity. Endogenous molecules areinherently non-toxic and may not yield toxic photoproducts afterphotoradiation.

Table 1 lists some photoactivatable molecules capable of beingphotoactivated to induce an auto vaccine effect.

TABLE 1 SSET and TTET rate constants for bichromophoric peptidesK_(SSET)(s⁻¹) R_(model)(Å) Compound λ_(ex) (nm) E_(SSET) k_(s) of donor(s⁻¹) k_(SSET) (s⁻¹) (Average) R₀ (Å) R (Å) (Average) E_(TTET) k_(TTET)(s⁻¹) 1B 224 96.3 9.5 × 10⁵ 2.44 × 10⁸ 1.87 × 10⁸ 14.7 9 9.5 266 95  1.8× 10⁸ 2.5   5 × 10² 280 94 1.36 × 10⁸ 1A 224 80 9.5 × 10⁶  3.8 × 10⁷3.67 × 10⁷ 14.7 11.8 14.1 266 79  3.6 × 10⁷ 2 3.6 × 10² 280 79  3.6 ×10⁷ 2B 224 77 9.5 × 10⁵  3.1 × 10⁷  3.9 × 10⁷ 14.7 11.9 5.5 266 81  3.9× 10⁷ 32 9.4 × 10³ 280 83  4.7 × 10⁷ 2A 224 69 9.5 × 10⁵  2.1 × 10⁷   3× 10⁷ 14.7 12.2 8.1 74.3 5.7 × 10⁴ 266 80  3.7 × 10⁷ 280 77  3.2 × 10⁷1A

1B

2A

2B

Table 2 lists some additional endogenous photoactivatable molecules.

TABLE 2 Biocompatible, endogenous fluorophore emitters. Excitation Max.Emission Max. Endogenous Fluorophores (nm) (nm) Amino acids: Tryptophan280 350 Tyrosine 275 300 Phenylalanine 280 280 Structural Proteins:Collagen 325, 360 400, 405 Elastin 290, 325 340, 400 Enzymes andCoenzymes: flavin adenine dinucleotide 450 535 reduced nicotinamidedinucelotide 290, 351 440, 460 reduced nicotinamide dinucelotidephosphate 336 464 Vitamins: Vitamins A 327 510 Vitamins K 335 480Vitamins D 390 480 Vitamins B₆ compounds: Pyridoxine 332, 340 400Pyridoxamine 335 400 Pyridoxal 330 385 Pyridoxic acid 315 425 Pyridoxalphosphate   5′-330 400 Vitamin B₁₂ 275 305 Lipids: Phospholipids 436540, 560 Lipofuscin 340-395 540, 430-460 Ceroid 340-395 430-460, 540Porphyrins 400-450 630, 690

The nature of the predetermined cellular change will depend on thedesired pharmaceutical outcome. Exemplary cellular changes may include,but are not limited to, apoptosis, necrosis, up-regulation of certaingenes, down-regulation of certain genes, secretion of cytokines,alteration of cytokine receptor responses, or a combination thereof.

As used herein, an “energy modulation agent” refers to an agent that iscapable of receiving an energy input from a source and then re-emittinga different energy to a receiving target. Energy transfer amongmolecules may occur in a number of ways. The form of energy may beelectronic, thermal, electromagnetic, kinetic, or chemical in nature.The energy can be modulated up to emit higher energy from the energymodulation agent compared to the input initiation energy, or can bemodulated down to emit lower energy from the energy modulation agentcompared to the input initiation energy. Energy may be transferred fromone molecule to another (intermolecular transfer) or from one part of amolecule to another part of the same molecule (intramolecular transfer).For example, a modulation agent may receive electromagnetic energy andre-emit the energy in the form of thermal energy. In preferredembodiments, the energy modulation agent receives higher energy (e.g.x-ray) and re-emits in lower energy (e.g. UV-A). Energy transferprocesses are also referred to as molecular excitation. Some modulationagents may have a very short energy retention time (on the order offs-ns, e.g. fluorescent molecules) whereas others may have a very longhalf-life (on the order of seconds to hours, e.g. luminescent inorganicmolecules or phosphorescent molecules). Suitable energy modulationagents include, but are not limited to, a biocompatible metalnanoparticle, metal coated with a biocompatible outer layer, achemiluminescent molecule whose rate of luminescence is increased bymicrowave activation, fluorescing dye molecule, gold nanoparticle, awater soluble quantum dot encapsulated by polyamidoamine dendrimers, aluciferase, a biocompatible phosphorescent molecule, a biocompatiblefluorescent molecule, a biocompatible scattering molecule, a combinedelectromagnetic energy harvester molecule, and a lanthanide chelatecapable of intense luminescence. Various exemplary uses of these aredescribed below in preferred embodiments.

The modulation agents may further be coupled to a carrier for cellulartargeting purposes. For example, a biocompatible molecule, such as afluorescing metal nanoparticle or fluorescing dye molecule that emits inthe UV-A band, may be selected as the energy modulation agent.

The energy modulation agent may be preferably directed to the desiredsite (e.g. a tumor) by systemic administration to a subject. Forexample, a UV-A emitting energy modulation agent may be concentrated inthe tumor site by physical insertion or by conjugating the UV-A emittingenergy modulation agent with a tumor specific carrier, such as anantibody, nucleic acid, peptide, a lipid, chitin or chitin-derivative, achelate, a surface cell receptor, molecular imprints, aptamers, or otherfunctionalized carrier that is capable of concentrating the UV-Aemitting source in a specific target tumor.

Additionally, the energy modulation agent can be used alone or as aseries of two or more energy modulation agents wherein the energymodulation agents provide an energy cascade. Thus, the first energymodulation agent in the cascade will absorb the activation energy,convert it to a different energy which is then absorbed by the secondenergy modulation in the cascade, and so forth until the end of thecascade is reached with the final energy modulation agent in the cascadeemitting the energy necessary to activate the activatable pharmaceuticalagent.

Although the activatable pharmaceutical agent and the energy modulationagent can be distinct and separate, it will be understood that the twoagents need not be independent and separate entities. In fact, the twoagents may be associated with each other via a number of differentconfigurations. Where the two agents are independent and separatelymovable from each other, they generally interact with each other viadiffusion and chance encounters within a common surrounding medium.Where the activatable pharmaceutical agent and the energy modulationagent are not separate, they may be combined into one single entity.

The initiation energy source can be any energy source capable ofproviding energy at a level sufficient to activate the activatable agentdirectly, or to provide the energy modulation agent with the inputneeded to emit the activation energy for the activatable agent (indirectactivation). Preferable initiation energy sources include, but are notlimited to, UV-A lamps or fiber optic lines, a light needle, anendoscope, and a linear accelerator that generates x-ray, gamma-ray, orelectron beams. The energy used can be any type, including but notlimited to, gamma ray, x-ray, UV, near-UV, visible, Near IR, IR,microwave, radio wave, etc. In a preferred embodiment the initiationenergy capable of penetrating completely through the subject. Within thecontext of the present invention, the phrase “capable of penetratingcompletely through the subject” is used to refer to energy that canpenetrate to any depth within the subject to activate the activatablepharmaceutical agent. It is not required that the any of the energyapplied actually pass completely through the subject, merely that it becapable of doing so in order to permit penetration to any desired depthto activate the activatable pharmaceutical agent. Exemplary initiationenergy sources that are capable of penetrating completely through thesubject include, but are not limited to, x-rays, gamma rays, electronbeams, microwaves and radio waves.

In one embodiment, the source of the initiation energy can be aradiowave emitting nanotube, such as those described by K. Jensen, J.Weldon, H. Garcia, and A. Zettl in the Department of Physics at theUniversity of California at Berkeley (seehttp://socrates.berkeley.edu/˜argon/nanoradio/radio.html, the entirecontents of which are hereby incorporated by reference). These nanotubescan be administered to the subject, and preferably would be coupled tothe activatable pharmaceutical agent or the energy modulation agent, orboth, such that upon application of the initiation energy, the nanotubeswould accept the initiation energy (preferably radiowaves), then emitradiowaves in close proximity to the activatable pharmaceutical agent,or in close proximity to the energy modulation agent, to then causeactivation of the activatable pharmaceutical agent. In such anembodiment, the nanotubes would act essentially as a radiowave focusingor amplification device in close proximity to the activatablepharmaceutical agent or energy modulation agent.

Alternatively, the energy emitting source may be an energy modulationagent that emits energy in a form suitable for absorption by a furtherenergy modulation agent. For example, the initiation energy source maybe acoustic energy and one energy modulation agent may be capable ofreceiving acoustic energy and emitting photonic energy (e.g.sonoluminescent molecules) to be received by another energy modulationagent that is capable of receiving photonic energy. Other examplesinclude transfer agents that receive energy at x-ray wavelength and emitenergy at UV wavelength, preferably at UV-A wavelength. As noted above,a plurality of such energy modulation agents may be used to form acascade to transfer energy from initiation energy source via a series ofenergy modulation agents to activate the activatable agent.

Signal transduction schemes as a drug delivery vehicle may beadvantageously developed by careful modeling of the cascade eventscoupled with metabolic pathway knowledge to sequentially orsimultaneously activate multiple activatable pharmaceutical agents toachieve multiple-point alterations in cellular function.

Photoactivatable agents may be stimulated by an energy source, such asirradiation, resonance energy transfer, exciton migration, electroninjection, or chemical reaction, to an activated energy state that iscapable of effecting the predetermined cellular change desired. In apreferred embodiment, the photoactivatable agent, upon activation, bindsto DNA or RNA or other structures in a cell. The activated energy stateof the agent is capable of causing damage to cells, inducing apoptosis.The mechanism of apoptosis is associated with an enhanced immuneresponse that reduces the growth rate of cell proliferation disordersand may shrink solid tumors, depending on the state of the patient'simmune system, concentration of the agent in the tumor, sensitivity ofthe agent to stimulation, and length of stimulation.

A preferred method of treating a cell proliferation disorder of thepresent invention administers a photoactivatable agent to a patient,stimulates the photoactivatable agent to induce cell damage, andgenerates an auto vaccine effect. In one further preferred embodiment,the photoactivatable agent is stimulated via a resonance energytransfer.

One advantage is that multiple wavelengths of emitted radiation may beused to selectively stimulate one or more photoactivatable agents orenergy modulation agents capable of stimulating the one or morephotoactivatable agents. The energy modulation agent is preferablystimulated at a wavelength and energy that causes little or no damage tohealthy cells, with the energy from one or more energy modulation agentsbeing transferred, such as by Foerster Resonance Energy Transfer, to thephotoactivatable agents that damage the cell and cause the onset of thedesired cellular change, such as apoptosis of the cells.

Another advantage is that side effects can be greatly reduced bylimiting the production of free radicals, singlet oxygen, hydroxides andother highly reactive groups that are known to damage healthy cells.Furthermore, additional additives, such as antioxidants, may be used tofurther reduce undesired effects of irradiation.

Resonance Energy Transfer (RET) is an energy transfer mechanism betweentwo molecules having overlapping emission and absorption bands.Electromagnetic emitters are capable of converting an arrivingwavelength to a longer wavelength. For example, UV-B energy absorbed bya first molecule may be transferred by a dipole-dipole interaction to aUV-A-emitting molecule in close proximity to the UV-B-absorbingmolecule. Alternatively, a material absorbing a shorter wavelength maybe chosen to provide RET to a non-emitting molecule that has anoverlapping absorption band with the transferring molecule's emissionband. Alternatively, phosphorescence, chemiluminescence, orbioluminescence may be used to transfer energy to a photoactivatablemolecule. Alternatively, one can administer the initiation energy sourceto the subject. Within the context of the present invention, theadministering of the initiation energy source means the administrationof an agent, that itself produces the initiation energy, in a mannerthat permits the agent to arrive at the target cell within the subjectwithout being surgically inserted into the subject. The administrationcan take any form, including, but not limited to, oral, intravenous,intraperitoneal, inhalation, etc. Further, the initiation energy sourcein this embodiment can be in any form, including, but not limited to,tablet, powder, liquid solution, liquid suspension, liquid dispersion,gas or vapor, etc. In this embodiment, the initiation energy sourceincludes, but is not limited to, chemical energy sources, nanoemitters,nanochips, and other nanomachines that produce and emit energy of adesired frequency. Recent advances in nanotechnology have providedexamples of various devices that are nanoscale and produce or emitenergy, such as the Molecular Switch (or Mol-Switch) work by Dr. KeithFirman of the EC Research and Development Project, or the work ofCornell et al. (1997) who describe the construction of nanomachinesbased around ion-channel switches only 1.5 nm in size, which use ionchannels formed in an artificial membrane by two gramicidin molecules:one in the lower layer of the membrane attached to a gold electrode andone in the upper layer tethered to biological receptors such asantibodies or nucleotides. When the receptor captures a target moleculeor cell, the ion channel is broken, its conductivity drops, and thebiochemical signal is converted into an electrical signal. Thesenanodevices could also be coupled with the present invention to providetargeting of the target cell, to deliver the initiation energy sourcedirectly at the desired site. In another embodiment, the presentinvention includes the administration of the activatable pharmaceuticalagent, along with administration of a source of chemical energy such aschemiluminescence, phosphorescence or bioluminescence. The source ofchemical energy can be a chemical reaction between two or morecompounds, or can be induced by activating a chemiluminescent,phosphorescent or bioluminescent compound with an appropriate activationenergy, either outside the subject or inside the subject, with thechemiluminescence, phosphorescence or bioluminescence being allowed toactivate the activatable pharmaceutical agent in vivo afteradministration. The administration of the activatable pharmaceuticalagent and the source of chemical energy can be performed sequentially inany order or can be performed simultaneously. In the case of certainsources of such chemical energy, the administration of the chemicalenergy source can be performed after activation outside the subject,with the lifetime of the emission of the energy being up to severalhours for certain types of phosphorescent materials for example. Thereare no known previous efforts to use resonance energy transfer of anykind to activate an intercalator to bind DNA.

In the PEPST embodiment of the present invention, the present inventionis significantly different from the phototherapy technique oftenreferred to as Photo-thermal Therapy (PTT). To illustrate the differencebetween the present invention PEPST, a form of photospectral therapy(PST) and the PTT technique, the photochemical processes involved in PSTand PPT is discussed below.

When drug molecules absorb excitation light, electrons undergotransitions from the ground state to an excited electronic state. Theelectronic excitation energy subsequently relaxes via radiative emission(luminescence) and radiationless decay channels. When a molecule absorbsexcitation energy, it is elevated from S_(o) to some vibrational levelof one of the excited singlet states, S_(n), in the manifold S₁, . . . ,S_(n). In condensed media (tissue), the molecules in the S_(n) statedeactivate rapidly, within 10⁻¹³ to 10⁻¹¹ s via vibrational relaxation(VR) processes, ensuring that they are in the lowest vibrational levelsof S_(n) possible. Since the VR process is faster than electronictransitions, any excess vibrational energy is rapidly lost as themolecules are deactivated to lower vibronic levels of the correspondingexcited electronic state. This excess VR energy is released as thermalenergy to the surrounding medium. From the S_(n) state, the moleculedeactivates rapidly to the isoenergetic vibrational level of a lowerelectronic state such as S_(n-1) via an internal conversion (IC)process. IC processes are transitions between states of the samemultiplicity. The molecule subsequently deactivates to the lowestvibronic levels of S_(n-1) via a VR process. By a succession of ICprocesses immediately followed by VR processes, the molecule deactivatesrapidly to the ground state S₁. This process results in excess VR and ICenergy released as thermal energy to the surrounding medium leading tothe overheating of the local environment surrounding the light absorbingdrug molecules. The heat produced results in local cell or tissuedestruction. The light absorbing species include natural chromophores intissue or exogenous dye compounds such as indocyanine green,naphthalocyanines, and porphyrins coordinated with transition metals andmetallic nanoparticles and nanoshells of metals. Natural chromophores,however, suffer from very low absorption. The choice of the exogenousphotothermal agents is made on the basis of their strong absorptioncross sections and highly efficient light-to-heat conversion. Thisfeature greatly minimizes the amount of laser energy needed to inducelocal damage of the diseased cells, making the therapy method lessinvasive. A problem associated with the use of dye molecules is theirphotobleaching under laser irradiation. Therefore, nanoparticles such asgold nanoparticles and nanoshells have recently been used. The promisingrole of nanoshells in photothermal therapy of tumors has beendemonstrated [Hirsch, L. R., Stafford, R. J., Bankson, J. A., Sershen,S. R., Rivera, B., Price, R. E., Hazle, J. D., Halas, N. J., and West,J. L., Nanoshell-mediated near-infrared thermal therapy of tumors undermagnetic resonance guidance. PNAS, 2003. 100(23): p. 13549-13554]. Theuse of plasmonics-enhanced photothermal properties of metalnanoparticles for photothermal therapy has also been reviewed (XiaohuaHuang & Prashant K Jain & Ivan H. El-Sayed & Mostafa A. El-Sayed,“Plasmonic photothermal therapy (PPTT) using gold nanoparticles”, Lasersin Medical Science, August 2007)

The PST method of the present invention, however, is based on theradiative processes (fluorescence, phosphorescence, luminescence, Raman,etc) whereas the PTT method is based on the radiationless processes (IC,VR and heat conversion) in molecules.

Basic Principle of Plasmonics and Enhanced Electromagnetic Fields

Whereas the photothermal properties of plasmonics metal nanoparticleshave been used, the spectroscopic absorption and emission ofplasmonics-active nanoparticles in phototherapy have not been reported.

In the present invention PEPST, the plasmonics-enhanced spectroscopicproperties (spectral absorption, emission, scattering) are the majorfactors involved in the treatment.

The PEPST principle is based on the enhancement mechanisms of theelectromagnetic field effect. There are two main sources ofelectromagnetic enhancement: (1) first, the laser electromagnetic fieldis enhanced due to the addition of a field caused by the polarization ofthe metal particle; (2) in addition to the enhancement of the excitationlaser field, there is also another enhancement due to the moleculeradiating an amplified emission (luminescence, Raman, etc.) field, whichfurther polarizes the metal particle, thereby acting as an antenna tofurther amplify the Raman/Luminescence signal.

Electromagnetic enhancements are divided into two main classes: a)enhancements that occur only in the presence of a radiation field, andb) enhancements that occur even without a radiation field. The firstclass of enhancements is further divided into several processes. Plasmaresonances on the substrate surfaces, also called surface plasmons,provide a major contribution to electromagnetic enhancement. Aneffective type of plasmonics-active substrate comprises nanostructuredmetal particles, protrusions, or rough surfaces of metallic materials.Incident light irradiating these surfaces excites conduction electronsin the metal, and induces excitation of surface plasmons leading toRaman/luminescence enhancement. At the plasmon frequency, the metalnanoparticles (or nanostructured roughness) become polarized, resultingin large field-induced polarizations and thus large local fields on thesurface. These local fields increase the luminescence/Raman emissionintensity, which is proportional to the square of the applied field atthe molecule. As a result, the effective electromagnetic fieldexperienced by the analyte molecule on these surfaces is much largerthan the actual applied field. This field decreases as 1/r³ away fromthe surface. Therefore, in the electromagnetic models, theluminescence/Raman-active analyte molecule is not required to be incontact with the metallic surface but can be located anywhere within therange of the enhanced local field, which can polarize this molecule. Thedipole oscillating at the wavelength λ of Raman or luminescence can, inturn, polarize the metallic nanostructures and, if λ is in resonancewith the localized surface plasmons, the nanostructures can enhance theobserved emission light (Raman or luminescence).

There are two main sources of electromagnetic enhancement: (1) first,the laser electromagnetic field is enhanced due to the addition of afield caused by the polarization of the metal particle; (2) in additionto the enhancement of the excitation laser field, there is also anotherenhancement due to the molecule radiating an amplifiedRaman/luminescence field, which further polarizes the metal particle,thereby acting as an antenna to further amplify the Raman/luminescencesignal. Plasmonics-active metal nanoparticles also exhibit stronglyenhanced visible and near-infrared light absorption, several orders ofmagnitude more intense compared to conventional laser phototherapyagents. The use of plasmonic nanoparticles as highly enhancedphotoabsorbing agents thus provides a selective and efficientphototherapy strategy. The tunability of the spectral properties of themetal nanoparticles and the biotargeting abilities of the plasmonicnanostructures make the PEPST method promising.

The present invention PEPST is based on several important mechanisms:

-   -   Increased absorption of the excitation light by the plasmonic        metal nanoparticles, resulting in enhanced photoactivation of        drug molecules    -   Increased absorption of the excitation light by the plasmonic        metal nanoparticles that serve as more efficient energy        modulation agent systems, yielding more light for increased        excitation of PA molecules    -   Increased absorption of the excitation light by the photoactive        drug system adsorbed on or near the plasmonic metal        nanoparticles    -   Increased light absorption of the energy modulation agent        molecules adsorbed on or near the metal nanoparticles    -   Amplified light emission from the energy modulation agent        molecules adsorbed on or near the metal nanoparticles    -   Increased absorption of emission light emitted from the energy        modulation agent by the PA molecule

One of several phenomena that can enhance the efficiency of lightemitted (Raman or luminescence) from molecules adsorbed or near a metalnanostructures Raman scatter is the surface-enhanced Raman scattering(SERS) effect. In 1984, the general applicability of SERS as ananalytical technique was first reported by one of the present inventors,and the possibility of SERS measurement for a variety of chemicalsincluding several homocyclic and heterocyclic polyaromatic compounds [T.Vo-Dinh, M Y K Hiromoto, G. M Begun and R. L. Moody, “Surface-enhancedRaman spectroscopy for trace organic analysis,” Anal. Chem., vol. 56,1667, 1984]. Extensive research has been devoted to understanding andmodeling the Raman enhancement in SERS since the mid 1980's. FIG. 1, forexample, shows the early work by Kerker modeling electromagnetic fieldenhancements for spherical silver nanoparticles and metallic nanoshellsaround dielectric cores as far back as 1984 [M. M. Kerker, Acc. Chem.Res., 17, 370 (1984)]. This figure shows the result of theoreticalcalculations of electromagnetic enhancements for isolated sphericalnanospheres and nanoshells at different excitation wavelengths. Theintensity of the normally weak Raman scattering process is increased byfactors as large as 10¹³ or 10¹⁵ for compounds adsorbed onto a SERSsubstrate, allowing for single-molecule detection. As a result of theelectromagnetic field enhancements produced near nanostructured metalsurfaces, nanoparticles have found increased use as fluorescence andRaman nanoprobes.

The theoretical models indicate that it is possible to tune the size ofthe nanoparticles and the nanoshells to the excitation wavelength.Experimental evidence suggests that the origin of the 10⁶- to 10¹⁵-foldRaman enhancement primarily arises from two mechanisms: a) anelectromagnetic “lightning rod” effect occurring near metal surfacestructures associated with large local fields caused by electromagneticresonances, often referred to as “surface plasmons”; and b) a chemicaleffect associated with direct energy transfer between the molecule andthe metal surface.

According to classical electromagnetic theory, electromagnetic fieldscan be locally amplified when light is incident on metal nanostructures.These field enhancements can be quite large (typically 10⁶- to 10⁷-fold,but up to 10¹⁵-fold enhancement at “hot spots”). When a nanostructuredmetallic surface is irradiated by an electromagnetic field (e.g., alaser beam), electrons within the conduction band begin to oscillate ata frequency equal to that of the incident light. These oscillatingelectrons, called “surface plasmons,” produce a secondary electric fieldwhich adds to the incident field. If these oscillating electrons arespatially confined, as is the case for isolated metallic nanospheres orroughened metallic surfaces (nanostructures), there is a characteristicfrequency (the plasmon frequency) at which there is a resonant responseof the collective oscillations to the incident field. This conditionyields intense localized field enhancements that can interact withmolecules on or near the metal surface. In an effect analogous to a“lightning rod,” secondary fields are typically most concentrated atpoints of high curvature on the roughened metal surface.

Design, Fabrication and Operation of PEPST Probes

FIG. 2 shows a number of the various embodiments of PEPST probes thatcan be designed:

-   -   (A) probe comprising PA molecules bound to a metal (gold)        nanoparticle;    -   (B) PA-containing nanoparticle covered with metal nanoparticles;    -   (C) Metal nanoparticle covered with PA nanocap;    -   (D) PA-containing nanoparticle covered with metal nanocap;    -   (E) Metal nanoparticle covered with PA nanoshell;    -   (F) PA-containing nanoparticle covered with metal nanoshell; and    -   (G) PA-containing nanoparticle covered with metal nanoshell with        protective coating layer.

A basic embodiment of the PEPST probe is shown in FIG. 2A. This probecomprises PA molecules bound to a metal (e.g., gold) nanoparticle. FIG.3 illustrates the plasmonics-enhancement effect of the PEPST probe. Thegold nanoparticles can serve as a drug delivery platform. Goldnanoparticles have been described as a novel technology in the field ofparticle-based tumor-targeted drug delivery [Giulio F. Paciotti andLonnie Myer, David Weinreich, Dan Goia, Nicolae Pavel, Richard E.McLaughlin, Lawrence Tamarkin, “Colloidal Gold: A Novel NanoparticleVector for Tumor Directed Drug Delivery, Drug Delivery, 11:169-183,2004]. Particle delivery systems capable of escaping phagocyticclearance by the reticuloendothelial system (RES) can facilitatetargeting cancer therapeutics to solid tumors. Such delivery systemscould preferentially accumulate within the tumor microenvironment underideal conditions. A particle delivery system capable of sequestering aphototherapeutic drug selectively within a tumor may also reduce theaccumulation of the drug in healthy organs. Consequently, these deliverysystems may increase the relative efficacy or safety of therapy (lessradiation energy and intensity), and therefore, will increase the drug'stherapeutic efficiency.

Radiation of suitable energy is used to excite the PA drug molecules(e.g., aminolevulinic acid (ALA), porphyrins) and make them photoactive.For example, with the PDT drug ALA, light of a HeNe laser (632.8-nmexcitation) can be used for excitation. In this case the metalnanoparticles are designed to exhibit strong plasmon resonance bandaround 632.8 nm. The surface plasmon resonance effect amplifies theexcitation light at the nanoparticles, resulting in increasedphotoactivation of the PA drug molecules and improved therapyefficiency. The plasmonics-enhanced mechanism can also be used with theother PEPST probes in FIGS. 2B, 2C, 2D, 2E, 2F and 2G.

Structures of Plasmonics-Active Metal Nanostructures

Plasmon resonances arise within a metallic nanoparticle from thecollective oscillation of free electrons driven by an incident opticalfield. The plasmonic response of nanoparticles have played a role in agrowing number of applications, including surface-enhanced Ramanscattering (SERS), chemical sensing, drug delivery, photothermal cancertherapy and new photonic devices. The investigation and application ofplasmonics nanosubstrates for SERS detection has been used by one of thepresent inventors for over two decades [T. Vo-Dinh, “Surface-EnhancedRaman Spectroscopy Using Metallic Nanostructures,” Trends in Anal.Chem., 17, 557 (1998)]. The first report by one of the present inventorson the practical analytical use of the SERS techniques for traceanalysis of a variety of chemicals including several homocyclic andheterocyclic polyaromatic compounds was in 1984 [T. Vo-Dinh, M. Y. KHiromoto, G. M Begun and R. L. Moody, “Surface-enhanced Ramanspectroscopy for trace organic analysis,” Anal. Chem., vol. 56, 1667,1984]. Since then, the development of SERS technologies for applicationsin chemical sensing, biological analysis and medical diagnostics hasbeen ongoing. The substrates involve nanoparticles and semi-nanoshellscomprising a layer of nanoparticles coated by a metal (such as silver)on one side (nanocaps or half-shells). Several groups have shown thatplasmon resonances of spherical shells can be tuned by controlling theshell thickness and aspect ratios of the nanoshell structures [M. MKerker, Acc. Chem. Res., 17, 370 (1984); J. B. Jackson, S. L. Westcott,L. R. Hirsch, J L. West and N. H. Halas, “Controlling the surfaceenhanced Raman effect via the nanoshell geometry,” Appl. Phys. Lett.,vol. 82, 257-259, 2003; S. J. Norton and T Vo-Dinh, “PlasmonicResonances of nanoshells of Spheroidal Shape”, IEEE Trans.Nanotechnology, 6, 627-638 (2007)]. These shells typically comprise ametallic layer over a dielectric core. In one embodiment of the presentinvention, these shells comprise spheroidal shells, since the plasmonresonances (both longitudinal and transverse modes) are influenced byboth shell thickness and aspect ratio. A number of researchers haveexamined the plasmonic response of the solid spheroidal particle intheir analysis of surface-enhanced Raman scattering, although thespheroidal shell appears not to have been investigated. The presentinvention also includes prolate and oblate spheroidal shells, which showsome interesting qualitative features in their plasmon resonances. Thespheroidal shell presents two degrees of freedom for tuning: the shellthickness and the shell aspect ratio [S. J Norton and T Vo-Dinh,“Plasmonic Resonances of Nanoshells of Spheroidal Shape”, IEEE Trans.Nanotechnology, 6, 627-638 (2007)].

FIG. 4 shows some of the various embodiments of plasmonics-activenanostructures that can be designed, and are preferred embodiments ofthe present invention:

-   -   (A) Metal nanoparticle;    -   (B) Dielectric nanoparticle core covered with metal nanocap;    -   (C) Spherical metal nanoshell covering dielectric spheroid core;    -   (D) Oblate metal nanoshell covering dielectric spheroid core;    -   (E) Metal nanoparticle core covered with dielectric nanoshell;    -   (F) Metal nanoshell with protective coating layer;    -   (G) Multi layer metal nanoshells covering dielectric spheroid        core;    -   (H) Multi-nanoparticle structures;    -   (I) Metal nanocube and nanotriangle/nanoprism; and    -   (J) Metal cylinder.        PEPST Probes with Remotely-Activated Drug Release

In a further embodiment of the present invention, the PA drug moleculescan be incorporated into a material (e.g., biocompatible polymer) thatcan form a nanocap onto the metal (gold) nanoparticles. The material canbe a gel or biocompatible polymer that can have long-term continuousdrug release properties. Suitable gel or biocompatible polymers include,but are not limited to poly(esters) based on polylactide (PLA),polyglycolide (PGA), polycarpolactone (PCL), and their copolymers, aswell as poly(hydroxyalkanoate)s of the PHB-PHV class, additionalpoly(ester)s, natural polymers, particularly, modifiedpoly(saccharide)s, e.g., starch, cellulose, and chitosan, polyethyleneoxides, poly(ether)(ester) block copolymers, and ethylene vinyl acetatecopolymers. The drug release mechanism can also be triggered bynon-invasive techniques, such as RF, MW, ultrasound, photon (FIG. 5).

FIG. 6 shows other possible embodiments where the PA drug molecule isbound to the metal nanoparticles via a linker that can be cut by aphoton radiation. Such a linker includes, but is not limited to, abiochemical bond (FIG. 6A), a DNA bond (FIG. 6B), or an antibody-antigenbond (FIG. 6C). In another embodiment, the linker is a chemically labilebond that will be broken by the chemical environment inside the cell.These types of probes are useful for therapy modalities where the PAmolecules have to enter the nucleus (e.g., psoralen molecules need toenter the nucleus of cells and intercalate onto DNA). Since it is moredifficult for metal nanoparticles to enter the cell nucleus than forsmaller molecules, it is desirable to PEPST probes that have releasablePA molecules.

Disease-Targeted PEPST Probes

Aggregation of metal (such as silver or gold) nanoparticles(nanopsheres, nanorods, etc) is often a problem, especially withcitrate-capped gold nanospheres, cetyl trimethylammonium bromide(CTAB)-capped gold nanospheres and nanorods and nanoshells because theyhave poor stability when they are dispersed in buffer solution due tothe aggregating effect of salt ions. The biocompatibility can beimproved and nanoparticle aggregation prevented by capping thenanoparticles with polyethylene glycol (PEG) (by conjugation ofthiol-functionalized PEG with metal nanoparticles). Furthermore,PEGylated nanoparticles are preferentially accumulated into tumortissues due to the enhanced permeability and retention effect, known asthe “EPR” effect [Maedaa H, Fanga J. Inutsukaa T. Kitamoto Y (2003)Vascular permeability enhancement in solid tumor: various factors,mechanisms involved and its implications. Int Immunopharmacol 3:319-328;Paciotti G F, Myer L, Weinreich D, Goia D, Pavel N. McLaughlin R E,Tamarkin L (2004) Colloidal gold: a novel nanoparticles vector for tumordirected drug delivery. Drug Deliv 11:169-183]. Blood vessels in tumortissue are more “leaky” than in normal tissue, and as a result,particles, or large macromolecular species or polymeric speciespreferentially extravasate into tumor tissue. Particles and largemolecules tend to stay a longer time in tumor tissue due to thedecreased lymphatic system, whereas they are rapidly cleared out innormal tissue. This tumor targeting strategy is often referred to aspassive targeting whereas the antibody-targeting strategy is calledactive targeting.

To specifically target diseased cells, specific genes or proteinmarkers, the drug systems of the present invention can be bound to abioreceptor (e.g., antibody, synthetic molecular imprint systems, DNA,proteins, lipids, cell-surface receptors, aptamers, etc.).Immunotargeting modalities to deliver PA agents selectively to thediseased cells and tissue provide efficient strategies to achievingspecificity, minimizing nonspecific injury to healthy cells, andreducing the radiation intensity used. Biofunctionalization of metalnanoparticles (e.g., gold, silver) can be performed using commonlydeveloped and widely used procedures. There are several targetingstrategies that can be used in the present invention: (a) nanoparticlesconjugated to antibodies that recognize biomarkers specific to thediseased cells; (b) nanoparticles passivated by poly(ethylene) glycol(PEG), which is used to increase the biocompatibility and biostabilityof nanoparticles and impart them an increased blood retention time.

PEPST Probes with Bioreceptors

Bioreceptors are the key to specificity for targeting disease cells,mutated genes or specific biomarkers. They are responsible for bindingthe biotarget of interest to the drug system for therapy. Thesebioreceptors can take many forms and the different bioreceptors thathave been used are as numerous as the different analytes that have beenmonitored using biosensors. However, bioreceptors can generally beclassified into five different major categories. These categoriesinclude: 1) antibody/antigen, 2) enzymes, 3) nucleic acids/DNA, 4)cellular structures/cells and 5) biomimetic. FIG. 7 illustrates a numberof embodiments of the various PEPST probes with bioreceptors that can bedesigned. The probes are similar to those in FIG. 2 but have also abioreceptor for tumor targeting.

Antibody Probes. Antibody based targeting is highly active, specific andefficient. The antibodies are selected to target a specific tumor marker(e.g., anti-epidermal growth factor receptor (EGFR) antibodies targetedagainst overexpressed EGFR on oral and cervical cancer cells; anti-Her2antibodies against overexpressed Her2 on breast cancer cells) Antibodiesare biological molecules that exhibit very specific binding capabilitiesfor specific structures. This is very important due to the complexnature of most biological systems. An antibody is a complex biomolecule,made up of hundreds of individual amino acids arranged in a highlyordered sequence. For an immune response to be produced against aparticular molecule, a certain molecular size and complexity arenecessary: proteins with molecular weights greater then 5000 Da aregenerally immunogenic. The way in which an antigen and itsantigen-specific antibody interact may be understood as analogous to alock and key fit, by which specific geometrical configurations of aunique key enables it to open a lock. In the same way, anantigen-specific antibody “fits” its unique antigen in a highly specificmanner. This unique property of antibodies is the key to theirusefulness in immunosensors where only the specific analyte of interest,the antigen, fits into the antibody binding site.

DNA Probes. The operation of gene probes is based on the hybridizationprocess. Hybridization involves the joining of a single strand ofnucleic acid with a complementary probe sequence. Hybridization of anucleic acid probe to DNA biotargets (e.g., gene sequences of amutation, etc) offers a very high degree of accuracy for identifying DNAsequences complementary to that of the probe. Nucleic acid strands tendto be paired to their complements in the corresponding double-strandedstructure. Therefore, a single-stranded DNA molecule will seek out itscomplement in a complex mixture of DNA containing large numbers of othernucleic acid molecules. Hence, nucleic acid probe (i.e., gene probe)detection methods are very specific to DNA sequences. Factors affectingthe hybridization or reassociation of two complementary DNA strandsinclude temperature, contact time, salt concentration, and the degree ofmismatch between the base pairs, and the length and concentration of thetarget and probe sequences.

Biologically active DNA probes can be directly or indirectly immobilizedonto a drug system, such as the energy modulation agent system (e.g.,gold nanoparticle, a semiconductor, quantum dot, a glass/quartznanoparticles, etc.) surface to ensure optimal contact and maximumbinding. When immobilized onto gold nanoparticles, the gene probes arestabilized and, therefore, can be reused repetitively. Several methodscan be used to bind DNA to different supports. The method commonly usedfor binding DNA to glass involves silanization of the glass surfacefollowed by activation with carbodiimide or glutaraldehyde. Thesilanization methods have been used for binding to glass surfaces using3 glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxysilane(APTS), followed by covalently linking DNA via amino linkersincorporated either at the 3′ or 5′ end of the molecule during DNAsynthesis.

Enzyme Probes. Enzymes are often chosen as bioreceptors based on theirspecific binding capabilities as well as their catalytic activity. Inbiocatalytic recognition mechanisms, the detection is amplified by areaction catalyzed by macromolecules called biocatalysts. With theexception of a small group of catalytic ribonucleic acid molecules, allenzymes are proteins. Some enzymes require no chemical groups other thantheir amino acid residues for activity. Others require an additionalchemical component called a cofactor, which may be either one or moreinorganic ions, such as Fe²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, or a more complexorganic or metalloorganic molecule called a coenzyme. The catalyticactivity provided by enzymes allows for much lower limits of detectionthan would be obtained with common binding techniques. The catalyticactivity of enzymes depends upon the integrity of their native proteinconformation. If an enzyme is denatured, dissociated into its subunits,or broken down into its component amino acids, its catalytic activity isdestroyed. Enzyme-coupled receptors can also be used to modify therecognition mechanisms.

PEGylated-Vectors for PEPST Probes

The synthesis of these particles was first reported by Michael Faraday,who, in 1857, described the chemical process for the production ofnanosized particles of Au0 from gold chloride and sodium citrate(Faraday 1857). Initial formulations of the vector, manufactured bybinding only TNF to the particles, were less toxic than native TNF andeffective in reducing tumor burden in a murine model. Subsequent studiesrevealed that the safety of this vector was primarily due to its rapiduptake and clearance in the RES. This vector was reformulated to includemolecules of thiol-derivatized polyethylene glycol (PEG-THIOL) that werebound with molecules of TNF on the gold nanoparticles surface. The newvector, PT-cAu-TNF, avoids detection and clearance by the RES, andactively and specifically sequesters TNF within a solid tumor. Thealtered biodistribution correlated to improvements. In the presentinvention, a preferred embodiment includes the use of PEGylated-Aunanoparticles-PA drug systems to avoid detection and clearance by theRES.

Immobilization of Biomolecules to Metal Nanoparticles

The immobilization of biomolecules (PA molecules, drugs, proteins,enzymes, antibodies, DNA, etc.) to a solid support can use a widevariety of methods published in the literature. Binding can be performedthrough covalent bonds taking advantage of reactive groups such as amine(—NH₂) or sulfide (—SH) that naturally are present or can beincorporated into the biomolecule structure. Amines can react withcarboxylic acid or ester moieties in high yield to form stable amidebonds. Thiols can participate in maleimide coupling, yielding stabledialkylsulfides.

A solid support of interest in the present invention is the metal(preferably gold or silver) nanoparticles. The majority ofimmobilization schemes involving metal surfaces, such as gold or silver,utilize a prior derivatization of the surface with alkylthiols, formingstable linkages. Alkylthiols readily form self-assembled monolayers(SAM) onto silver surfaces in micromolar concentrations. The terminus ofthe alkylthiol chain can be used to bind biomolecules, or can be easilymodified to do so. The length of the alkylthiol chain has been found tobe an important parameter, keeping the biomolecules away from thesurface, with lengths of the alkyl group from 4 to 20 carbons beingpreferred. For example, in the case for DNA hybridization this has beenshown to displace nonspecifically adsorbed HS-(CH2)-6-ss-DNA andreorient chemically attached HS-(CH2)-6-ss-DNA in such a way to make themajority of surface bound probes accessible for hybridization (M. Culha,D. L. Stokes, an dT. Vo-Dinh, “Surface-Enhanced Raman Scattering forCancer Diagnostics: Detection of the BLC2 Gene,” Expert Rev. Mol.Diagnostics, 3, 669-675 (2003)). Furthermore, to avoid direct,non-specific DNA adsorption onto the surface, alkylthiols have been usedto block further access to the surface, allowing only covalentimmobilization through the linker [Steel, A. B.; Heme, T. M.; Tarlov, M.J. Anal. Chem. 1998, 70, 4670-7; Heme, T. M.; Tarlov, M. J. J. Am. Chem.Soc. 1997, 119, 8916-20]

There are many methods related to the preparation of stableoligonucleotide conjugates with gold particles by usingthiol-functionalized biomolecules that had previously been shown to formstrong gold-thiol bonds. Oligonucleotides with 5′-terminal alkanethiolfunctional groups as anchors can be bound to the surface of goldnanoparticles, and the resulting labels were robust and stable to bothhigh and low temperature conditions [R. Elghanian, J. J. Storhoff, R. C.Mucic, R. L. Letsinger and C. A. Mirkin, Selective colorimetricdetection of polynucleotides based on the distance-dependent opticalproperties of gold nanoparticles. Science 277 (1997), pp. 1078-1081]. Acyclic dithiane-epiandrosterone disulfide linker has been developed forbinding oligonucleotides to gold surfaces [R. Elghanian, J. J. Storhoff,R. C. Mucic, R. L. Letsinger and C. A. Mirkin, Selective colorimetricdetection of polynucleotides based on the distance-dependent opticalproperties of gold nanoparticles. Science 277 (1997), pp. 1078-1081]. Liet al. have reported a trithiol-capped oligonucleotide that canstabilize gold metal nanoparticles having diameters ≧100 nm, whileretaining hybridization properties that are comparable to acyclic ordithiol-oligonucleotide modified particles [Z. Li, R. C. Jin, C. A.Mirkin and R. L. Letsinger, Multiple thiol-anchor capped DNA-goldnanoparticle conjugates. Nucleic Acids Res. 30 (2002), pp. 1558-1562].

In general silver nanoparticles cannot be effectively passivated byalkylthiol-modified oligonucleotides using the established experimentalprotocols that were developed for gold particles. A method of generatingcore-shell particles comprising a core of silver and a thin shell ofgold has allowed silver nanoparticles to be readily functionalized withalkylthiol-oligonucleotides using the proven methods used to preparepure gold particle-oligonucleotide conjugates. [Y. W. Cao, R. Jin and C.A. Mirkin, DNA-modified core-shell Ag/Au nanoparticles. J. Am. Chem.Soc. 123 (2001), pp. 7961-7962].

To facilitate the use of biomolecule-conjugated plasmonics-activenanoprobes (PAN) it is important that the recognition region of thebiomolecule is fully accessible to the biotarget. Commonly apolynucleotide extension sequence is incorporated to serve as a spacerbetween the PAN and the oligonucleotide recognition region. To achievehigh sensitivity and selectivity in assays based on DNA hybridization itis important that the PAN label colloidal solution is stable. Recently,Storhoff et al. [J. J. Storhoff, R. Elghanian, C. A. Mirkin and R. L.Letsinger, Sequence-dependent stability of DNA-modified goldnanoparticles. Langmuir 18 (2002), pp. 6666-6670] have shown that thebase composition of the oligonucleotide has a significant effect oncolloid stability and on oligonucleotide surface coverage. Otsuka et al.have used a heterobifunctional thiol-PEG (polyethylene glycol)derivative as a linker to stabilize gold PRPs [H. Otsuka, Y. Akiyama, YNagasaki and K Kataoka, Quantitative and reversible lectin-inducedassociation of gold nanoparticles modified withα-lactosyl-ω-mercapto-poly(ethylene glycol). J. Am. Chem. Soc. 123(2001), pp. 8226-8230].

Proteins are usually bound to PANs using non-covalent, passiveabsorption. Alternatively, a mercapto-undecanoic acid linker/spacermolecule can be used to attach NeutrAvidin covalently to gold and silversegmented nanorods [I. D. Walton, S. M. Norton, A. Balasingham, L. He,D. F. Oviso, D. Gupta, P. A. Raju, M. J. Natan and R. G. Freeman,Particles for multiplexed analysis in solution: detection andidentification of striped metallic particles using optical microscopy.Anal. Chem. 74 (2002), pp. 2240-2247]. The thiol groups bind to themetal surface, and the carboxyl functional groups on the particlesurface are activated using EDC and s-NHS reagents and then cross-linkedto the amino groups in NeutrAvidin. The ability to fabricate core-shellparticles where the core is metal and the shell is composed of latex,silica, polystyrene or other non-metal material provides a promisingalternative approach to immobilizing biomolecules and engineeringparticle surfaces [T. K. Mandal, M. S. Fleming and D. R. Walt,Preparation of polymer coated gold nanoparticles by surface-confinedliving radical polymerization at ambient temperature. Nano Letters 2(2002), pp. 3-7; S. O. Obare, N. R. Jana and C. J Murphy, Preparation ofpolystyrene- and silica-coated gold nanorods and their use as templatesfor the synthesis of hollow nanotubes. Nano Letters 1 (2001), pp.601-603; C. Radloff and N. J. Halas, Enhanced thermal stability ofsilica-encapsulated metal nanoshells. Appl. Phys. Lett. 79 (2001), pp.674-676; L. Quaroni and G. Chumanov, Preparation of polymer-coatedfunctionalized silver nanoparticles. J. Am. Chem. Soc. 121 (1999), pp.10642-10643p; F. Caruso, Nanoengineering of particle surfaces. Adv.Mater. 13 (2001), pp. 11-22].

Silver surfaces have been found to exhibit controlled self-assemblykinetics when exposed to dilute ethanolic solutions of alkylthiols. Thetilt angle formed between the surface and the hydrocarbon tail rangesfrom 0 to 15°. There is also a larger thiol packing density on silver,when compared to gold [Burges, J. D.; Hawkridge, F. M. Langmuir 1997,13, 3781-6]. After self-assembled monolayer (SAM) formation ongold/silver nanoparticles, alkylthiols can be covalently coupled tobiomolecules. The majority of synthetic techniques for the covalentimmobilization of biomolecules utilize free amine groups of apolypeptide (enzymes, antibodies, antigens, etc) or of amino-labeled DNAstrands, to react with a carboxylic acid moiety forming amide bonds. Asa general rule, a more active intermediate (labile ester) is firstformed with the carboxylic acid moiety and in a later stage reacted withthe free amine, increasing the coupling yield. Successful couplingprocedures include, but are not limited to:

Binding Procedure Using N-Hydroxysuccinimide (NHS) and its Derivatives

The coupling approach involves the esterification under mild conditionsof a carboxylic acid with a labile group, an N-hydroxysuccinimide (NHS)derivative, and further reaction with free amine groups in a polypeptide(enzymes, antibodies, antigens, etc) or amine-labeled DNA, producing astable amide [Boncheva, M.; Scheibler, L.; Lincoln, P.; Vogel, H.;Akerman, B. Langmuir 1999, 15, 4317-20]. NHS reacts almost exclusivelywith primary amine groups. Covalent immobilization can be achieved in aslittle as 30 minutes. Since H₂O competes with —NH₂ in reactionsinvolving these very labile esters, it is important to consider thehydrolysis kinetics of the available esters used in this type ofcoupling. The derivative of NHS,O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,increases the coupling yield by utilizing a leaving group that isconverted to urea during the carboxylic acid activation, hence favorablyincreasing the negative enthalpy of the reaction.

Binding Procedure Using Maleimide

Maleimide can be used to immobilize biomolecules through available —SHmoieties. Coupling schemes with maleimide have been proven useful forthe site-specific immobilization of antibodies, Fab fragments, peptides,and SH-modified DNA strands. Sample preparation for the maleimidecoupling of a protein involves the simple reduction of disulfide bondsbetween two cysteine residues with a mild reducing agent, such asdithiothreitol, 2-mercaptoethanol or tris(2-carboxyethyl)phosphinehydrochloride. However, disulfide reduction will usually lead to theprotein losing its natural conformation, and might impair enzymaticactivity or antibody recognition. The modification of primary aminegroups with 2-iminothiolane hydrochloride (Traut's reagent) to introducesulfhydryl groups is an alternative for biomolecules lacking them. Freesulfhydryls are immobilized to the maleimide surface by an additionreaction to unsaturated carbon-carbon bonds [Jordan, C. E., et al.,1997].

Binding Procedure Using Carbodiimide.

Surfaces modified with mercaptoalkyldiols can be activated with1,1′-carbonyldiimidazole (CDI) to form a carbonylimidazole intermediate.A biomolecule with an available amine group displaces the imidazole toform a carbamate linkage to the alkylthiol tethered to the surface[Potyrailo, R. A., et al., 1998].

Other Experimental Procedures to Conjugate Biomolecules to Metal (e.g.,Silver, Gold) Nanoparticles.

In one preferred embodiment, nanoparticles of metal colloid hydrosolsare prepared by rapidly mixing a solution of AgNO₃ with ice-cold NaBH₄.For developing a SMP probes, a DNA segment is bound to a nanoparticle ofsilver or gold. The immobilization of biomolecules (e.g., DNA,antibodies, enzymes, etc.) to a solid support through covalent bondsusually takes advantage of reactive groups such as amine (—NH₂) orsulfide (—SH) that naturally are present or can be incorporated into thebiomolecule structure. Amines can react with carboxylic acid or estermoieties in high yield to form stable amide bonds. Thiols canparticipate in maleimide coupling yielding stable dialkylsulfides.

In one preferred embodiment, silver nanoparticles are used. In onepreferred embodiment, the immobilization schemes involving Ag surfacesutilize a prior derivatization of the surface with alkylthiols, formingstable linkages are used. Alkylthiols readily form self-assembledmonolayers (SAM) onto silver surfaces in micromolar concentrations. Theterminus of the alkylthiol chain can be directly used to bindbiomolecules, or can be easily modified to do so. The length of thealkylthiol chain was found to be an important parameter, keeping thebiomolecules away from the surface. Furthermore, to avoid direct,non-specific DNA adsorption onto the surface, alkylthiols were used toblock further access to the surface, allowing only covalentimmobilization through the linker.

Silver/gold surfaces have been found to exhibit controlled self-assemblykinetics when exposed to dilute ethanolic solutions of alkylthiols. Thetilt angle formed between the surface and the hydrocarbon tail rangesfrom 0 to 15°. There is also a larger thiol packing density on silver,when compared to gold.

After SAM formation on silver nanoparticles, alkylthiols can becovalently coupled to biomolecules. The majority of synthetic techniquesfor the covalent immobilization of biomolecules utilize free aminegroups of a polypeptide (enzymes, antibodies, antigens, etc) or ofamino-labeled DNA strands, to react with a carboxylic acid moietyforming amide bonds. In one embodiment, more active intermediate (labileester) is first formed with the carboxylic acid moiety and in a laterstage reacted with the free amine, increasing the coupling yield.Successful coupling procedures include:

The coupling approach used to bind DNA to a silver nanoparticle involvesthe esterification under mild conditions of a carboxylic acid with alabile group, an N-hydroxysuccinimide (NHS) derivative, and furtherreaction with free amine groups in a polypeptide (enzymes, antibodies,antigens, etc) or amine-labeled DNA, producing a stable amide [4]. NHSreacts almost exclusively with primary amine groups. Covalentimmobilization can be achieved in as little as 30 minutes. Since H₂Ocompetes with —NH₂ in reactions involving these very labile esters, itis important to consider the hydrolysis kinetics of the available estersused in this type of coupling. The derivative of NHS used in FIG. 101,O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,increases the coupling yield by utilizing a leaving group that isconverted to urea during the carboxylic acid activation, hence favorablyincreasing the negative enthalpy of the reaction.

Spectral Range of Light Used for PEPST

A plasmonics enhanced effect can occur throughout the electromagneticregion provided the suitable nanostructures, nanoscale dimensions, metaltypes are used. Therefore, the PEPST concept is valid for the entireelectromagnetic spectrum, i.e, energy, ranging from gamma rays and Xrays throughout ultraviolet, visible, infrared, microwave and radiofrequency energy. However, for practical reasons, visible and NIR lightare used for silver and gold nanoparticles, since the plasmon resonancesfor silver and gold occur in the visible and NIR region, respectively.Especially for gold nanoparticles, the NIR region is very appropriatefor non-invasive therapy.

Photon Excitation in the Therapeutic Window of Tissue

There are several methods using light to excite photoactivate compoundsnon-invasively. We can use light having wavelengths within the so-called“therapeutic window” (700-1300 nm). The ability of light to penetratetissues depends on absorption. Within the spectral range known as thetherapeutic window (or diagnostic window), most tissues are sufficientlyweak absorbers to permit significant penetration of light. This windowextends from 600 to 1300 nm, from the orange/red region of the visiblespectrum into the NIR. At the short-wavelength end, the window is boundby the absorption of hemoglobin, in both its oxygenated and deoxygenatedforms. The absorption of oxygenated hemoglobin increases approximatelytwo orders of magnitude as the wavelength shortens in the region around600 nm. At shorter wavelengths many more absorbing biomolecules becomeimportant, including DNA and the amino acids tryptophan and tyrosine. Atthe infrared (IR) end of the window, penetration is limited by theabsorption properties of water. Within the therapeutic window,scattering is dominant over absorption, and so the propagating lightbecomes diffuse, although not necessarily entering into the diffusionlimit. FIG. 8 shows a diagram of the therapeutic window of tissue. Thefollowing section discusses the use of one-photon and multi-photontechniques for therapy.

Light Excitation Methods: Single-Photon and Multi-Photon Excitation

Two methods can be used, one-photon or multi-photon excitation. If thetwo-photon technique is used, one can excite the PA molecules with lightat 700-1000 nm, which can penetrate deep inside tissue, in order toexcite molecules that absorb in the 350-500 nm spectral region. Thisapproach can excite the psoralen compounds, which absorb in the 290-350nm spectral region and emit in the visible. With the one-photon method,the photo-activator (PA) drug molecules can directly absorb excitationlight at 600-1300 nm. In this case we can design a psoralen-relatedsystem (e.g., psoralens having additional aromatic rings or otherconjugation to alter the ability to absorb at different wavelengths) oruse other PA systems: photodynamic therapy drugs, ALA, etc.

PEPST Modality for Photopheresis Using X ray Excitation Need for X-RayExcitation

Photopheresis has been demonstrated to be an effective treatment for anumber of diseases. However, there is a strong need to developnon-invasive modalities where the excitation light can directlyirradiate the photoactive compounds without the need for removal andreinfusion of blood from patients. One method for an improved andpractical modality for such therapy was described in U.S. Ser. No.11/935,655, filed Nov. 6, 2007, the entire contents of which are herebyincorporated by reference.

Although X-ray can excite compounds in deep tissue non-invasively, X-rayis not easily absorbed by organic drug compounds. The present inventionprovides a solution to that problem, by the providing of a molecularsystem that can absorb the X-ray energy and change that energy intoother energies that can be used to activate drug molecules. Morespecifically, the molecular system that can absorb and change the X-rayenergy in the present invention is the PEPST probes comprisingnanoparticles.

In this embodiment, the present invention uses X-rays for excitation.The advantage is the ability to excite molecules non-invasively sinceX-ray can penetrate deep in tissue. However, the limitation is the factthat X-ray does not interact with most molecules. In one embodiment ofthe present invention, the drug molecule (or PA) is bound to a molecularentity, referred to as an “energy modulation agent” that can interactwith the X-rays, and then emit light that can be absorbed by the PA drugmolecules. (FIG. 9)

PEPST Probes for X Ray Excitation

In the previous sections, the advantage of gold nanoparticles asplasmonics-active systems have been discussed. Furthermore, goldnanoparticles are also good energy modulation agent systems since theyare biocompatible and have been shown to be a possible candidate forcontrast agents for X-ray [Hainfeld et al, The British Journal ofradiology, 79, 248, 2006]. The concept of using high-Z materials fordose enhancement in cancer radiotherapy was advanced over 20 years ago.The use of gold nanoparticles as a dose enhancer seems more promisingthan the earlier attempts using microspheres and other materials for twoprimary reasons. First, gold has a higher Z number than iodine (I, Z=53)or gadolinium (Gd, Z=64), while showing little toxicity, up to at least3% by weight, on either the rodent or human tumour cells. The goldnanoparticles were non-toxic to mice and were largely cleared from thebody through the kidneys. This novel use of small gold nanoparticlespermitted achievement of the high metal content in tumours necessary forsignificant high-Z radioenhancement [James F Hainfeld, Daniel N Slatkinand Henry M Smilowitz, The use of gold nanoparticles to enhanceradiotherapy in mice, Phys. Med. Biol. 49 (2004)]

Delivering a lethal dose of radiation to a tumor while minimizingradiation exposure of nearby normal tissues remains the greatestchallenge in radiation therapy. The dose delivered to a tumour duringphoton-based radiation therapy can be enhanced by loading high atomicnumber (Z) materials such as gold (Au, Z=79) into the tumor, resultingin greater photoelectric absorption within the tumor than in surroundingtissues. Thus, gold clearly leads to a higher tumor dose than eitheriodine or gadolinium. Second, nanoparticles provide a better mechanismthan microspheres, in terms of delivering high-Z materials to the tumor,overcoming some of the difficulties found during an earlier attemptusing gold microspheres [Sang Hyun Cho, Estimation of tumour doseenhancement due to gold nanoparticles during typical radiationtreatments: a preliminary Monte Carlo study, Phys. Med. Biol. 50 (2005)]

Gold (or metal) complexes with PA ligands: Gold (or metal) complexeswith PA can preferably be used in the present invention. The metal canbe used as an energy modulation agent system. For example, goldcomplexes with psoralen-related ligands can be used as a hybrid energymodulation agent-PA system. The gold molecules serve as the energymodulation agent system and the ligand molecules serve as the PA drugsystem. Previous studies indicated that gold(I) complexes withdiphosphine and bipyridine ligands exhibit X-ray excited luminescence[Ref. 3: Kim et al, Inorg. Chem., 46, 949, 2007].

FIG. 10 shows a number of the various embodiments of PEPST probes thatcan be preferably used for X ray excitation of energy modulationagent-PA system. These probes comprise:

-   -   (A) PA molecules bound to energy modulation agent and to        plasmonic metal nanoparticle;    -   (B) Plasmonic metal nanoparticle with energy modulation agent        nanocap covered with PA molecules;    -   (C) PA-covered nanoparticle with plasmonic metal nanoparticles;    -   (D) Energy modulation agent-containing nanoparticle covered with        PA molecules and plasmonic metal nanocap;    -   (E) Plasmonic metal nanoparticle core with energy modulation        agent nanoshell covered with PA molecule; and    -   (F) PA molecule bound to energy modulation agent (attached to        plasmonics metal nanoparticle) nanoparticle by detachable        biochemical bond.

Examples of PEPST System Based on Energy Modulation Agent-PA

For purposes of simplification, the following discussion is centered ongold as the metal material and CdS as the energy modulation agentmaterial (which can also be used as DNA stabilized CdS, see Ma et al,Langmuir, 23 (26), 12783-12787 (2007)) and psoralen as the PA molecule.However, it is to be understood that many other embodiments of metalmaterial, energy modulation agent and PA molecule are possible withinthe bounds of the present invention, and the following discussion is forexemplary purposes only. Suitable metals that can be used in plasmonresonating shells or other plasmon resonating structures can be include,but are not limited to, gold, silver, platinum, palladium, nickel,ruthenium, rhenium, copper, gallium, and cobalt.

In the embodiment of FIG. 10A, the PEPST system comprises goldnanoparticles, an energy modulation agent nanoparticle (e.g., CdS)linked to a PA drug molecule (e.g., psoralen). X ray is irradiated toCdS, which absorbs X rays [Hua et al, Rev. Sci. Instrum., 73, 1379,2002] and emits CdS XEOL light (at 350-400 nm) that isplasmonics-enhanced by the gold nanoparticle. This enhanced XEOL lightis used to photoactivate psoralen (PA molecule). In this case thenanostructure of the gold nanoparticle is designed to enhance the XEOLlight at 350-400 nm.

In the embodiment of FIG. 10B, the PEPST system comprises aplasmonics-active metal (gold) nanoparticle with energy modulation agentnanocap (CdS) covered with PA molecules (e.g., psoralen). X ray isirradiated to CdS, which absorbs X ray and emits XEOL light that isplasmonics-enhanced by the gold nanoparticle. This enhanced XEOL lightis used to photoactivate psoralen (PA molecule).

In the embodiment of FIG. 10C, the PEPST system comprises a PA (e.g.,psoralen)-covered CdS nanoparticle with smaller plasmonic metal (gold)nanoparticles. X ray is irradiated to CdS, which absorbs X ray and emitsXEOL light that is plasmonics-enhanced by the gold nanoparticle. Thisenhanced XEOL light is used to photoactivate psoralen (PA molecule).

In the embodiment of FIG. 10D, the energy modulation agent corecomprises CdS or CsCl nanoparticles covered with a nanocap of gold. Xray is irradiated to CdS or CsCl, which absorbs X ray [[Jaegle et al, J.Appl. Phys., 81, 2406, 1997] and emits XEOL light that isplasmonics-enhanced by the gold nanocap structure. This enhanced XEOLlight is used to photoactivate psoralen (PA molecule).

Similarly, the embodiment in FIG. 10E comprises a spherical gold corecovered by a shell of CdS or CsCl. X ray is irradiated to CdS or CsClmaterial, which absorbs X ray [Jaegle et al, J. Appl. Phys., 81, 2406,1997] and emits XEOL light that is plasmonics-enhanced by the goldnanosphere. This enhanced XEOL light is used to photoactivate psoralen(PA molecule).

In the embodiment of FIG. 10F, the PEPST system comprises goldnanoparticles, and an energy modulation agent nanoparticle (e.g., CdS)linked to a PA drug molecule (e.g., psoralen) by a link that can bedetached by radiation. X ray is irradiated to CdS, which absorbs X rayand emits CdS XEOL light (at 350-400 nm) that is plasmonics-enhanced bythe gold nanoparticle. This enhanced XEOL light is used to photoactivatepsoralen (PA molecule). In this case the nanostructure of the goldnanoparticle is designed to enhance the XEOL light at 350-400 nm.

In alternative embodiments, the metal nanoparticles or single nanoshellsare replaced by multi layers of nanoshells [Kun Chen, Yang Liu,Guillermo Ameer, Vadim Backman, Optimal design of structured nanospheresfor ultrasharp light-scattering resonances as molecular imagingmultilabels, Journal of Biomedical Optics, 10(2), 024005 (March/April2005)].

In other alternative embodiments the metal nanoparticles are coveredwith a layer (1-30 nm) of dielectric material (e.g. silica). Thedielectric layer (or nanoshell) is designed to prevent quenching of theluminescence light emitted by the energy modulation agent (also referredto as EEC) molecule(s) due to direct contact of the metal with theenergy modulation agent molecules. In yet other alternative embodiments,the energy modulation agent molecules or materials are bound to (or inproximity of) a metal nanoparticle via a spacer (linker). The spacer isdesigned to prevent quenching of the luminescence light emitted by theenergy modulation agent molecules or materials.

Other Useable Materials

The energy modulation agent materials can include any materials that canabsorb X ray and emit light in order to excite the PA molecule. Theenergy modulation agent materials include, but are not limited to:

metals (gold, silver, etc);

quantum dots;

semiconductor materials;

scintillation and phosphor materials;

materials that exhibit X-ray excited luminescence (XEOL);

organic solids, metal complexes, inorganic solids, crystals, rare earthmaterials (lanthanides), polymers, scintillators, phosphor materials,etc.; and materials that exhibit excitonic properties.

Quantum dots, semiconductor nanostructures. Various materials related toquantum dots, semiconductor materials, etc. can be used as energymodulation agent systems. For example CdS-related nanostructures havebeen shown to exhibit X-ray excited luminescence in the UV-visibleregion [Hua et al, Rev. Sci. Instrum., 73, 1379, 2002].

Scintillator Materials as energy modulation agent systems. Variousscintillator materials can be used as energy modulation agents sincethey absorb X-ray and emit luminescence emission, which can be used toexcite the PA system. For example, single crystals of molybdates can beexcited by X-ray and emit luminescence around 400 nm [Mirkhin et al,Nuclear Instrum. Meth. In Physics Res. A, 486, 295 (2002].

Solid Materials as energy modulation agent systems: Various solidmaterials can be used as energy modulation agents due to their X-rayexcited luminescence properties. For example CdS (or CsCl) exhibitluminescence when excited by soft X-ray [Jaegle et al, J. Appl. Phys.,81, 2406, 1997].

XEOL materials: lanthanides or rare earth materials and their oxides,such as Y₂O₃, which can also contain one or more dopants [L. Soderholm,G. K Liu, Mark R. Antonioc, F. W. Lytle, X-ray excited opticalluminescence .XEOL. detection of x-ray absorption fine structure .XAFZ,J. Chem. Phys, 109, 6745, 1998], Masashi Ishiia, Yoshihito Tanaka andTetsuya Ishikawa, Shuji Komuro and Takitaro Morikawa, Yoshinobu Aoyagi,Site-selective x-ray absorption fine structure analysis of an opticallyactive center in Er-doped semiconductor thin film using x-ray-excitedoptical luminescence, Appl. Phys. Lett, 78, 183, 2001]

Some examples of metal complexes exhibiting XEOL which can be used asenergy modulation agent systems are shown in FIGS. 11 and 12. Suchstructures can be modified by replacing the metal atom with metalnanoparticles in order to fabricate a plasmonics-enhance PEPST probe. Inthe present invention, the experimental parameters including size, shapeand metal type of the nano structure can be selected based upon theexcitation radiation (NIR or X ray excitation), the photoactivationradiation (UVB), and/or the emission process from the energy modulationagent system (visible NIR).

U.S. Pat. No. 7,008,559 (the entire contents of which are incorporatedherein by reference) describes the upconversion performance of ZnS whereexcitation at 767 nm produces emission in the visible range. Thematerials described in U.S. Pat. No. 7,008,559 (including the ZnS aswell as Er³⁺ doped BaTiO₃ nanoparticles and Yb³⁺ doped CsMnCl₃) aresuitable in various embodiments of the invention.

Further materials suitable as energy modulation agents include, but arenot limited to, CdTe, CdSe, ZnO, CdS, Y₂O₃, MgS, CaS, SrS and BaS. Suchmaterials may be any semiconductor and more specifically, but not by wayof limitation, sulfide, telluride, selenide, and oxide semiconductorsand their nanoparticles, such as Zn_(1-x)Mn_(x)S_(y),Zn_(1-x)Mn_(x)Se_(y), Zn_(1-x)Mn_(x)Te_(y), Cd_(1-x)MnS_(y),Cd_(1-x)Mn_(x)Se_(y), Cd_(1-x)Mn_(x)Te_(y), Pb_(1-x)Mn_(x)S_(y),Pb_(1-x)Mn_(x)Se_(y), Pb_(1-x)Mn_(x)Te_(y), Mg_(1-x)MnS_(y),Ca_(1-x)Mn_(x)S_(y), Ba_(1-x)Mn_(x)S_(y) and Sr_(1-x), etc. (wherein,0<x≦1, and 0<y≦1). Complex compounds of the above-describedsemiconductors are also contemplated for use in the invention—e.g.(M_(1-z)N_(z))_(1-x)Mn_(x)A_(1-y)B_(y) (M=Zn, Cd, Pb, Ca, Ba, Sr, Mg;N=Zn, Cd, Pb, Ca, Ba, Sr, Mg; A=S, Se, Te, O; B=S, Se, Te, O; 0<x≦1,0<y≦1, 0<z≦1). Two examples of such complex compounds areZn_(0.4)Cd_(0.4)Mn_(0.2)S and Zn_(0.9)Mn_(0.1)S_(0.8)Se_(0.2).Additional energy modulation materials include insulating andnonconducting materials such as BaF₂, BaFBr, and BaTiO₃, to name but afew exemplary compounds. Transition and rare earth ion co-dopedsemiconductors suitable for the invention include sulfide, telluride,selenide and oxide semiconductors and their nanoparticles, such as ZnS;Mn; Er; ZnSe; Mn, Er; MgS; Mn, Er; CaS; Mn, Er; ZnS; Mn, Yb; ZnSe; Mn,Yb; MgS; Mn, Yb; CaS; Mn, Yb etc., and their complex compounds:(M_(1-z)N_(z))_(1-x)(Mn_(q)R_(1-q))_(x)A_(1-y)B_(y) (M=Zn, Cd, Pb, Ca,Ba, Sr, Mg; N=Zn, Cd, Pb, Ca, Ba, Sr, Mg; A=S, Se, Te, 0; B=S, . . .0<z≦1, o<q≦1).

Some nanoparticles such as ZnS:Tb³⁺, Er³⁺; ZnS:Tb³⁺; Y₂O₃:Tb³⁺;Y₂O₃:Tb³⁺, Er³⁺; ZnS:Mn²⁺; ZnS:Mn, Er³⁺ are known in the art to functionfor both down-conversion luminescence and upconversion luminescence, andcan thus be used in various embodiments of the present invention.

Principle of Plasmonics-Enhancement Effect of the PEPST Probe UsingX-Ray Excitation

One embodiment of the basic PEPST probe embodiment comprises PAmolecules bound to an energy modulation agent and to plasmonic metal(gold) nanoparticles. First the metal nanoparticle can serve as a drugdelivery platform (see previous discussion). Secondly, the metalnanoparticle can play 2 roles:

(1) Enhancement of the X-ray electromagnetic field

(2) Enhancement of the emission signal of the energy modulation agentsystem.

The X ray radiation, used to excite the energy modulation agent system,is amplified by the metal nanoparticle due to plasmon resonance. As aresult the energy modulation agent system exhibits more emission lightthat is used to photoactivate the PA drug molecules (e.g., psoralens)and make them photoactive. In this case the metal nanoparticles aredesigned to exhibit strong plasmon resonance at or near the X raywavelengths. The surface plasmon resonance effect amplifies theexcitation light at the nanoparticles, resulting in increasedphotoactivation of the PA drug molecules and improved therapyefficiency. The plasmonics-enhanced mechanism can also be used with theother PEPST probes described above.

FIG. 13 illustrates the plasmonics-enhancement effect of the PEPSTprobe. X-ray used in medical diagnostic imaging has photon energies fromapproximately 10 to 150 keV, which is equivalent to wavelengths rangefrom 1.2 to 0.0083 Angstroms. [λ (Angstrom)=12.4/E (keV)]. Soft X raycan go to 10 nm. The dimension of plasmonics-active nanoparticlesusually have dimensions on the order or less than the wavelengths of theradiation used. Note that the approximate atomic radius of gold isapproximately 0.15 nanometers. At the limit, for gold the smallest“nanoparticle” size is 0.14 nm (only 1 gold atom). A nanoparticle withsize in the hundreds of nm will have approximately 10⁶-10⁷ gold atoms.Therefore, the range of gold nanoparticles discussed in this inventioncan range from 1-10⁷ gold atoms.

The gold nanoparticles can also enhance the energy modulation agentemission signal, which is use to excite the PA molecule. For psoralens,this spectral range is in the UVB region (320-400 nm). Silver or goldnanoparticles, nanoshell and nanocaps have been fabricated to exhibitstrong plasmon resonance in this region. FIG. 14 shows excitation andemission fluorescence spectra of a psoralen compound(8-methoxypsoralen).

PEPST Energy Modulation Agent-PA Probe with Detachable PA.

Some photoactive drugs require that the PA molecule to enter thenucleus. FIG. 15 shows an embodiment of a PEPST probe where the PA drugmolecule is bound to the metal nanoparticles via a linker (FIG. 15A)that can be cut by photon radiation (FIG. 155B). Such a probe is usefulfor therapy modalities where the PA molecules have to enter the nucleus,e.g., psoralen molecules need to enter the nucleus of cells andintercalate onto DNA (FIG. 15C). Since it is more difficult for metalnanoparticles to enter the cell nucleus than for smaller molecules, itis preferable to use PEPST probes that have releasable PA molecules.

Suitable linkers for linking the PA drug molecule to the metalnanoparticles include, but are not limited to, labile chemical bondsthat can be broken by remote energy excitation (from outside the body,e.g., MW, IR, photoacoustic energy, ultrasound energy, etc.), labilechemical bonds that can be broken by the chemical environment insidecells, antibody-antigen, nucleic acid linkers, biotin-streptavidin, etc.

Nanoparticle Chain for Dual Plasmonics Effect

As discussed previously, there is the need to develop nanoparticlesystems that can have dual (or multi) plasmonics resonance modes. FIG.16 illustrates an embodiment of the present invention PEPST probe havinga chain of metal particles having different sizes and coupled to eachother, which could exhibit such dual plasmonics-based enhancement. Forexample the parameters (size, metal type, structure, etc) of the largernanoparticle (FIG. 16, left) can be tuned to NIR, VIS or UV light whilethe smaller particle (FIG. 16, right) can be tuned to X ray. There isalso a coupling effect between these particles.

These nanoparticle chains are useful in providing plasmonics enhancementof both the incident radiation used (for example, x-ray activation ofCdS) as well as plasmonics enhancement of the emitted radiation thatwill then activate the PA. Similar nanoparticles systems have been usedas nanolens [Self-Similar Chain of Metal Nanospheres as an EfficientNanolens, Kuiru Li, Mark I. Stockman, and David J. Bergman, PhysicalReview Letter, VOLUME 91, NUMBER 22, 227402-1, 2003].

Drug Delivery Platforms

Liposome Delivery of Energy Modulation Agent-PA Systems

The field of particle-based drug delivery is currently focused on twochemically distinct colloidal particles, liposomes and biodegradablepolymers. Both delivery systems encapsulate the active drug. The drug isreleased from the particle as it lyses, in the case of liposomes, ordisintegrates, as described for biodegradable polymers. One embodimentof the present invention uses liposomal delivery of energy modulationagent-PA systems (e.g., gold nanoshells) for therapy. An exemplaryembodiment is described below, but is not intended to be limiting to thespecific lipids, nanoparticles or other components recited, but ismerely for exemplary purposes:

Preparation of Liposomes. the Liposome Preparation Method is Adaptedfrom Hölig et. al Hölig, P., Bach, M., Völkel, T., Nahde, T., Hoffmann,S., Müller, R., and Kontermann, R. E., Novel RGD lipopeptides for thetargeting of liposomes to integrin-expressing endothelial and melanomacells. Protein Engineering Design and Selection, 2004. 17(5): p.433-441]. Briefly, the lipids PEG-DPPE, PC, and Rh-DPPE are mixed inchloroform in a round bottom flask and evaporated (Hieroglyph RotaryEvaporator, Rose Scientific Ltd., Edmonton, Alberta, Canada) toeliminate chloroform. The dry film is dehydrated into aqueous phase withusing PBS solution. A dry lipid film is prepared by rotary evaporationfrom a mixture of PC, cholesterol, and PEG-DPPE and then hydrated intoaqueous phase using PBS. The mixture is vigorously mixed by overtaxingand bath solicited (Instrument, Company) and the suspension extrudedthrough polycarbonate filter using Liposofast apparatus (Avestin Inc.,Ottawa, ON, Canada) (pore-size 0.8 μm). Preparation of liposomes isperformed as follows; 0.1 mmol of PC is dispersed in 8 ml of chloroformand supplemented with 0.5 mol of PEG-DPPE in 20 ml of chloroform. 0.3mmol rhodamine-labeled phosphatidylethanolamine (Rh-DPPE) is thenincorporated into the liposomes. The organic solvents are then removedby rotary evaporation at 35° C. for 2 h leaving a dry lipid film. Goldnanoshells are encapsulated into liposomes by adding them to the PBShydration buffer and successively into the dry lipid film. This mixtureis emulsified in a temperature controlled sonicator for 30 minutes at35° C. followed by vortexing for 5 min. Encapsulated gold nanoshells areseparated from unencapsulated gold nanoshells by gentle centrifugationfor 5 minutes at 2400 r.p.m (1200 g). The resulting multilamellarvesicles suspension is extruded through polycarbonate filter usingLiposofast apparatus (Avestin Inc., Ottawa, ON, Canada) (pore-size 0.8μm). The aqueous mixture is obtained and stored at 4° C.

Fabrication of Gold Nanoparticles: The Frens method [Frens, G.,Controlled nucleation for the regulation of the particle size inmonodisperse gold solutions. Nature (London) Phys Sci, 1973. 241: p.20-22] can be used in the present invention to synthesize a solution ofgold nanoparticles ranging in diameter from 8-10 nm. Briefly, 5.0×10⁻⁶mol of HAuCl₄ is dissolved in 19 ml of deionized water producing a faintyellowish solution. This solution is heated with vigorous stirring in arotary evaporator for 45 minutes. 1 ml of 0.5% sodium citrate solutionis added and the solution is stirred for an additional 30 minutes. Thecolor of the solution gradually changed from the initial faint yellowishto clear, grey, purple and finally a tantalizing wine-red color similarto merlot. The sodium citrate used serves in a dual capacity, firstacting as a reducing agent, and second, producing negative citrate ionsthat are adsorbed onto the gold nanoparticles introducing surface chargethat repels the particles and preventing nanocluster formation.

Preparation and Internalization of Liposome-encapsulated GoldNanoshells: Liposome-encapsulated gold nanoshells are incubated withMCF-7 cells grown on partitioned cover-slips for intracellular delivery.This is done by adding 10 μl of liposome-encapsulated gold nanoshellsper 1 ml of cell culture medium. This is incubated for 30 minutes in ahumidified (86% RH) incubator at 37° C. and 5% CO₂. This cell is usedfor localization studies; to track the rhodamine-DPPE-labeled liposomesinto the cytoplasm of the MCF-7 cell. After incubation, the cells grownon cover-slips are washed three times in cold PBS and fixed using 3.7%formaldehyde in PBS. Rhodamine staining by rhodamine-DPPE-labeledliposomes is analyzed using a Nikon Diaphot 300 inverted microscope(Nikon, Inc., Melville, N.Y.).

Non-Invasive Cleavage of the Drug System In Vivo

After delivery of the drug system into the cell, there is sometimes theneed to have the PA system (e.g. psoralen) in the nucleus in order tointeract with DNA. If the PA is still linked to the energy modulationagent, both of them have to be transported into the nucleus. In the casewith gold nanoparticles as the energy modulation agent system, there areseveral methods to incubate cells in vitro. For in vivo applications,one can link the PA to the gold nanoparticles using a chemical linkagethat can be released (or cut) using non-invasive methods such asinfrared, microwave, or ultrasound waves. An example of linkage isthrough a chemical bond or through a bioreceptor, such as an antibody.In this case, the PA is the antigen molecule bound to the energymodulation agent system that has an antibody targeted to the PA.

When the energy modulation agent-Ab-PA enters the cell, the PA moleculescan be released from the energy modulation agent Ab system. To releasethe PA molecule from the antibody, chemical reagents can be used tocleave the binding between antibody and antigen, thus regenerating thebiosensor [Vo-Dinh et al, 1988]. This chemical procedure is simple butis not practical inside a cell due to possible denaturation of the cellby the chemical. In previous studies, it has been demonstrated that thegentle but effective MHz-range ultrasound has the capability to releaseantigen molecules from the antibody-energy modulation agent system[Moreno-Bondi, M., Mobley, J., and Vo-Dinh, T., “RegenerableAntibody-based Biosensor for Breast Cancer,” J. Biomedical Optics, 5,350-354 (2000)]. Thus, an alternative embodiment is to use gentleultrasonic radiation (non-invasively) to remove the PA (antigen) fromthe antibody at the energy modulation agent system.

In a preferred embodiment, the PA molecule is bound to the energymodulation agent by a chemically labile bond [Jon A. Wolff, and David B.Rozema, Breaking the Bonds: Non-viral Vectors Become Chemically Dynamic,Molecular Therapy (2007) 16(1), 8-15]. A promising method of improvingthe efficacy of this approach is to create synthetic vehicles (SVs) thatare chemically dynamic, so that delivery is enabled by the cleavage ofchemical bonds upon exposure to various physiological environments orexternal stimuli. An example of this approach is the use of maskedendosomolytic agents (MEAs) that improve the release of nucleic acidsfrom endosomes, a key step during transport. When the MEA enters theacidic environment of the endosome, a pH-labile bond is broken,releasing the agent's endosomolytic capability.

Use of Ferritin and Apoferritin as Targeted Drug Delivery

Another embodiment to deliver the energy modulation agent-PA drugsinvolves the use of ferritin and apoferritin compounds. There isincreasing interest in ligand-receptor-mediated delivery systems due totheir non-immunogenic and site-specific targeting potential to theligand-specific bio-sites. Platinum anticancer drug have beenencapsulated in apoferritin [Zhen Yang, Xiaoyong Wang, Huajia Diao,Junfeng Zhang, Hongyan Li, Hongzhe Sun and Zijian Guo, Encapsulation ofplatinum anticancer drugs by apoferritin, Chem. Commun. 33, 2007,3453-3455]. Ferritin, the principal iron storage molecule in a widevariety of organisms, can also be used as a vehicle for targeted drugdelivery. It contains a hollow protein shell, apoferritin, which cancontain up to its own weight of hydrous ferric oxide-phosphate as amicrocrystalline micelle. The 24 subunits of ferritin assembleautomatically to form a hollow protein cage with internal and externaldiameters of 8 and 12 nm, respectively. Eight hydrophilic channels ofabout 0.4 nm, formed at the intersections of subunits, penetrate theprotein shell and lead to the protein cavity. A variety of species suchas gadolinium (Gd³⁺) contrast agents, desferrioxamine B, metal ions, andnanoparticles of iron salts can be accommodated in the cage ofapoferritin. Various metals such as iron, nickel, chromium and othermaterials have been incorporated into apoferritin [Iron incorporationinto apoferritin. The role of apoferritin as a ferroxidase, The Journalof Biological Chemistry [002]-9258] Bakker yr:1986 vol:261 iss:28pg:13182-5; Mitsuhiro Okuda¹, Kenji Iwahori², Ichiro Yamashita²,Hideyuki Yoshimura¹*, Fabrication of nickel and chromium nanoparticlesusing the protein cage of apoferritin, Biotechnology Bioengineering,Volume 84, Issue 2, Pages 187-194]. Zinc selenide nanoparticles (ZnSeNPs) were synthesized in the cavity of the cage-shaped proteinapoferritin by designing a slow chemical reaction system, which employstetraaminezinc ion and selenourea. The chemical synthesis of ZnSe NPswas realized in a spatially selective manner from an aqueous solution,and ZnSe cores were formed in almost all apoferritin cavities withlittle bulk precipitation [Kenji Iwahori, Keiko Yoshizawa, MasahiroMuraoka, and Ichiro Yamashita, Fabrication of ZnSe Nanoparticles in theApoferritin Cavity by Designing a Slow Chemical Reaction System, Inorg.Chem., 44 (18), 6393-6400, 2005].

A simple method for synthesizing gold nanoparticles stabilized by horsespleen apoferritin (HSAF) is reported using NaBH₄ or3-(N-morpholino)propanesulfonic acid (MOPS) as the reducing agent [LeiZhang, Joe Swift, Christopher A. Butts, Vijay Yerubandi and Ivan J.Dmochowski, Structure and activity of apoferritin-stabilized goldnanoparticles, Journal of Inorganic Biochemistry, Vol. 101, 1719-1729,2007]. Gold sulfite (Au₂S) nanoparticles were prepared in the cavity ofthe cage-shaped protein, apoferritin. Apoferritin has a cavity, 7 nm indiameter, and the diameter of fabricated Au₂S nanoparticles is about thesame size with the cavity and size dispersion was small. [KeikoYoshizawa, Kenji Iwahori, Kenji Sugimoto and Ichiro Yamashita,Fabrication of Gold Sulfide Nanoparticles Using the Protein Cage ofApoferritin, Chemistry Letters, Vol. 35 (2006), No. 10 p. 1192]. Thus,in a preferred embodiment, the PA or energy modulation agent-PAcompounds are encapsulated inside the apoferrtin shells.

Use of Ferritin and Apoferritin as Enhanced Targeting Agents

It was reported that ferritin could be internalized by some tumortissues, and the internalization was associated with themembrane-specific receptors [S. Fargion, P. Arosio, A. L. Fracanzoni, V.Cislaghi, S. Levi, A. Cozzi, A Piperno and A. G. Firelli, Blood, 1988,71, 753-757; P.C. Adams, L. W. Powell and J. W. Halliday, Hepatology,1988, 8, 719-721]. Previous studies have shown that ferritin-bindingsites and the endocytosis of ferritin have been identified in neoplasticcells [M. S. Bretscher and J. N. Thomson, EMBO J, 1983, 2, 599-603].Ferritin receptors have the potential for use in the delivery ofanticancer drugs into the brain [S. W. Hulet, S. Powers and J. R.Connor, J. Neurol. Sci., 1999, 165, 48-55]. In one embodiment, thepresent invention uses ferritin or apoferritin to both encapsulate PAand energy modulation agent-PA systems and also target tumor cellsselectively for enhanced drug delivery and subsequent phototherapy. Inthis case no additional bioreactors are needed.

FIG. 17 schematically illustrates the use of encapsulated photoactiveagents (FIG. 17A) for delivery into tissue and subsequent release of thephotoactive drugs after the encapsulated systems enter the cell. Notethe encapsulated system can have a bioreceptor for selective tumortargeting (FIG. 17B). Once inside the cell, the capsule shell (e.g.,liposomes, apoferritin, etc.) can be broken (FIG. 17C) usingnon-invasive excitation (e.g., ultrasound, RF, microwave, IR, etc) inorder to release the photoactive molecules that can get into the nucleusand bind to DNA (FIG. 17D).

Non-Invasive Phototherapy Using PEPST Modality

FIG. 18 illustrates the basic operating principle of the PEPST modality.The PEPST photoactive drug molecules are given to a patient by oralingestion, skin application, or by intravenous injection. The PEPSTdrugs travel through the blood stream inside the body towards thetargeted tumor (either via passive or active targeting strategies). Ifthe disease is systematic in nature a photon radiation at suitablewavelengths is used to irradiate the skin of the patient, the lightbeing selected to penetrate deep inside tissue (e.g., NIR or X ray). Forsolid tumors, the radiation light source is directed at the tumor.Subsequently a treatment procedure can be initiated using delivery ofenergy into the tumor site. One or several light sources may be used asdescribed in the previous sections. One embodiment of therapy comprisessending NIR radiation using an NIR laser through focusing optics.Focused beams of other radiation types, including but not limited to Xray, microwave, radio waves, etc. can also be used and will depend uponthe treatment modalities used.

Exciton-Plasmon Enhanced Phototherapy (EPEP)

Basic Principle of Exciton-Induced Phototherapy

Excitons in Solid Materials

Excitons are often defined as “quasiparticles” inside a solid material.In solid materials, such as semiconductors, molecular crystals andconjugated organic materials, light excitation at suitable wavelength(such as X ray, UV and visible radiation, etc) can excite electrons fromthe valence band to the conduction band. Through the Coulombinteraction, this newly formed conduction electron is attracted, to thepositively charged hole it left behind in the valence band. As a result,the electron and hole together form a bound state called an exciton.(Note that this neutral bound complex is a “quasiparticle” that canbehave as a boson—a particle with integer spin which obeys Bose-Einsteinstatistics; when the temperature of a boson gas drops below a certainvalue, a large number of bosons ‘condense’ into a single quantumstate—this is a Bose-Einstein condensate (BEC). Exciton production isinvolved in X-ray excitation of a solid material. Wide band-gapmaterials are often employed for transformation of the x-ray toultraviolet/visible photons in the fabrication of scintillators andphosphors [Martin Nikl, Scintillation detectors for x-rays, Meas. Sci.Technol. 17 (2006) R37-R54]. The theory of excitons is well known inmaterials research and in the fabrication and applications ofsemiconductors and other materials. However, to the present inventors'knowledge, the use of excitons and the design of energy modulation agentmaterials based on exciton tunability for phototherapy have not beenreported.

During the initial conversion a multi-step interaction of a high-energyX-ray photon with the lattice of the scintillator material occursthrough the photoelectric effect and Compton scattering effect; forX-ray excitation below 100 keV photon energy the photoelectric effect isthe main process. Many excitons (i.e., electron-hole pairs) are producedand thermally distributed in the conduction bands (electrons) andvalence bands (holes). This first process occurs within less than 1 ps.In the subsequent transport process, the excitons migrate through thematerial where repeated trapping at defects may occur, leading to energylosses due to nonradiative recombination, etc. The final stage,luminescence, consists in consecutive trapping of the electron-holepairs at the luminescent centers and their radiative recombination. Theelectron-hole pairs can be trapped at the defects and recombine,producing luminescent. Luminescent dopants can also be used as traps forexciton.

Exciton Traps

Exciton traps can be produced using impurities in the crystal hostmatrix. In impure crystals with dipolar guest molecules the electrontrap states may arise when electron is localized on a neighbor of theimpurity molecule. Such traps have been observed in anthracene dopedwith carbazole [Kadshchuk, A. K, Ostapenko, N. I., Skryshevskii, Yu. A.,Sugakov, V. I. and Susokolova, T. O., Mol. Cryst. and Liq. Cryst., 201,167 (1991)]. The formation of these traps is due to the interaction ofthe dipole moment of the impurity with charge carrier. When theconcentration of the dopant (or impurities) is increased, spectraexhibit additional structure of spectrum due to the trapping of carrierson clusters of impurity molecules. Sometimes, impurities and dopants arenot required: the electron or exciton can also be trapped on astructural defect in such crystals due to the electrostatic interactionwith reoriented dipole moment of disturbed crystal molecules [S. V.Izvekov, V. I. Sugakov, Exciton and Electron Traps on Structural Defectsin Molecular Crystals with Dipolar Molecules, Physica Scripta. Vol. T66,255-257, 1996]. One can design structural defects in molecular crystalsthat serve as exiton traps. The development of GaAs/AlGaAsnanostructures and use of nanofabrication technologies can designengineered exciton traps with novel quantum mechanical properties inmaterials

Design, Fabrication and Operation of EIP Probes

FIG. 20 shows various embodiments of EIP probes that can be designed:

-   -   (A) probe comprising PA molecules bound (through a linker, which        can be fixed or detachable) to an energy modulation agent        particle that can produce excitons under radiative excitation at        a suitable wavelength (e.g., X-ray). In this preferred        embodiment, the energy modulation agent materials have        structural defects that serve as traps for excitons.    -   (B) probe comprising PA molecules bound (through a linker, which        can be fixed or detachable) to an energy modulation agent        particle that can produce excitons under radiative excitation at        a suitable wavelength (e.g., X-ray). In this preferred        embodiment, the energy modulation agent materials have        impurities or dopant molecules that serve as traps for excitons.

EIP Probes with Tunable Emission:

The embodiment in probes B provide the capability to tune the energyconversion from an X ray excitation source into a wavelength of interestto excite the PA molecules. In 1976, D'Silva et al demonstrated thatpolynuclear aromatic hydrocarbons (PAH) molecules doped in a frozenn-alkane solids could be excited by X-ray and produce luminescence atvisible wavelengths characteristics of their luminescence spectra. [A.P. D'Silva, G. J. Oestreich, and V. A. Fassel, X-ray excited opticalluminescence of polynuclear aromatic hydrocarbons, Anal. Chem.; 1976;48(6) pp 915-917]. Tunable EIP probes can be designed to contain suchluminescent dopants such as highly luminescent PAHs exhibitingluminescence emission in the range of 300-400 nm suitable to activatepsoralen. A preferred embodiment of the EIP with tunable emissioncomprises a solid matrix (semiconductors, glass, quartz, conjugatedpolymers, etc) doped with naphthalene, phenanthrene, pyrene or othercompounds exhibiting luminescence (fluorescence) in the 300-400 nm range[T Vo-Dinh, Multicomponent analysis by synchronous luminescencespectrometry, Anal. Chem.; 1978; 50(3) pp 396-401]. See FIG. 21. The EECmatrix could be a semiconductor material, preferably transparent atoptical wavelength of interest (excitation and emission).

Other dopant species such as rare earth materials can also be used asdopants. FIG. 22 shows the X ray excitation optical luminescence (XEOL)of Europium doped in a matrix of BaFBr, emitting at 370-420 nm. U.S.Patent Application Publication No. 2007/0063154 (hereby incorporated byreference) describes these and other nanocomposite materials (andmethods of making them) suitable for XEOL.

FIG. 23 shows various embodiments of EIP probes that can be designed:

(A) probe comprising PA molecules bound around the energy modulationagent particle or embedded in a shell around an energy modulation agentparticle that can produce excitons under radiative excitation at asuitable wavelength (e.g., X-ray). In this preferred embodiment, theenergy modulation agent materials has structural defects that serve astraps for excitons.

(B) probe comprising PA molecules bound around the energy modulationagent particle or embedded in a shell around an energy modulation agentparticle that can produce excitons under radiative excitation at asuitable wavelength (e.g., X-ray). In this preferred embodiment, theenergy modulation agent materials have impurities or dopant moleculesthat serve as traps for excitons.

Principle of Exciton-Plasmon Enhanced Phototherapy (EPEP)

There is recent interest in an advanced photophysical concept involvingquantum optical coupling between electronic states (excitons), photonsand enhanced electromagnetic fields (plasmons). Such a concept involvingcoupling between excitons and plasmons can be used to enhance aphototherapy modality, referred to as Exciton-Plasmon EnhancedPhototherapy (EPEP).

A fundamental key concept in photophysics is the formation of newquasiparticles from admixtures of strongly-coupled states. Such mixedstates can have unusual properties possessed by neither originalparticle. The coupling between excitons and plasmons can be either weakor strong. When the light-matter interaction cannot be considered as aperturbation, the system is in the strong coupling regime. Bellesa et alshowed a strong coupling between a surface plasmon (SP) mode and organicexcitons occurs; the organic semiconductor used is a concentratedcyanine dye in a polymer matrix deposited on a silver film [Ref: J.Bellessa, * C. Bonnand, and J. C. Plenet, J Mugnier, Strong Couplingbetween Surface Plasmons and Excitons in an Organic Semiconductor, Phys.Rev. Lett, 93 (3), 036404-1, 2004]. Govorov et al describe thephotophysical properties of excitons in hybrid complexes consisting ofsemiconductor and metal nanoparticles. The interaction betweenindividual nanoparticles can produce an enhancement or suppression ofemission. Enhanced emission comes from electric field amplified by theplasmon resonance, whereas emission suppression is a result of energytransfer from semiconductor to metal nanoparticles. [Alexander O.Govorov, *, † Garnett W. Bryant,; Wei Zhang, ‡ Timur Skeini, t JaebeomLee, § Nicholas A. Kotov, § Joseph M Slocik, | and Rajesh R. Naik|,Exciton-Plasmon Interaction and Hybrid Excitons in Semiconductor-MetalNanoparticle Assemblies, Nano Lett., Vol. 6, No. 5, 984, 2006]. Bondarevet al also described a theory for the interactions between excitonicstates and surface electromagnetic modes in small-diameter (<1 nm)semiconducting single-walled carbon nanotubes (CNs). [I. V. Bondarev, K.Tatur and L. M. Woods, Strong exciton-plasmon coupling in semiconductingcarbon nanotubes].

Fedutik et al reported about the synthesis and optical properties of acomposite metal-insulator-semiconductor nanowire system which consistsof a wet-chemically grown silver wire core surrounded by a SiO₂ shell ofcontrolled thickness, followed by an outer shell of highly luminescentCdSe nanocrystals [Yuri Fedutik, † Vasily Temnov, † Ulrike Woggon, †Elena Ustinovich, ‡ and Mikhail Artemyev* ‡, Exciton-Plasmon Interactionin a Composite Metal-Insulator-Semiconductor Nanowire System, J. Am.Chem. Soc., 129 (48), 14939-14945, 2007]. For a SiO₂ spacer thickness of˜15 nm, they observed an efficient excitation of surface plasmons byexcitonic emission of CdSe nanocrystals. For small d, well below 10 nm,the emission is strongly suppressed (PL quenching), in agreement withthe expected dominance of the dipole-dipole interaction with the dampedmirror dipole [G. W. Ford and W. H. Weber, Electromagnetic interactionsof molecules with metal surfaces,” Phys. Rep. 113, 195-287 (1984)]. Fornanowire lengths up to ˜10 μm, the compositemetal-insulator-semiconductor nanowires ((Ag)SiO₂)CdSe act as awaveguide for ID-surface plasmons at optical frequencies with efficientphoton out coupling at the nanowire tips, which is promising forefficient exciton-plasmon-photon conversion and surface plasmon guidingon a submicron scale in the visible spectral range.

Experiments on colloidal solutions of Ag nanoparticles covered withJ-aggregates demonstrated the possibility of using the strong scatteringcross section and the enhanced field associated with surface plasmon togenerate stimulated emission from J-aggregate excitons with very lowexcitation powers. [Gregory A. Wurtz, * Paul R. Evans, William Hendren,Ronald Atkinson, Wayne Dickson, Robert J Pollard, and Anatoly V Zayats,Molecular Plasmonics with Tunable Exciton-Plasmon Coupling Strength inJ-Aggregate Hybridized Au Nanorod Assemblies, Nano Lett., Vol. 7, No. 5,1297, 2007]. Their coupling to surface plasmons excitations thereforeprovides a particularly attractive approach for creating low-poweredoptical devices. This process can lead to efficient X-ray coupling forphototherapy. In addition, the coupling of J-aggregates with plasmonicsstructures presents genuine fundamental interest in the creation ofmixed plasmon-exciton states.

Design, Fabrication and Operation of EPEP Probes

FIG. 24 shows various embodiments of EPEP probes of the presentinvention showing the exciton-plasmon coupling:

-   -   (A) probe comprising a PA molecule or group of PA molecules        bound (through a linker, which can be fixed or detachable) to an        energy modulation agent particle that can produce excitons under        radiative excitation at a suitable wavelength (e.g., X-ray). The        energy modulation agent particle is bound to (or in proximity        of) a metal nanoparticle covered with a nanoshell of silica (or        other dielectric material). The silica layer (or nanoshell) (see        FIG. 24A and FIG. 24B; layer nanoshell in white between energy        modulation material and metal nanostructures) is designed to        prevent quenching of the luminescence light emitted by the        energy modulation agent particle excited by X-ray. The metal        nanoparticle (Au, Ag, etc) is designed to induce plasmons that        enhance the X ray excitation that subsequently leads to an        increase in the energy modulation agent light emission,        ultimately enhancing the efficiency of photoactivation, i.e.        phototherapy. The structure of the nanoparticle can also be        designed such that the plasmonics effect also enhances the        energy modulation agent emission light. These processes are due        to strong coupling between excitons (in the energy modulation        agent materials and plasmons in the metal nanoparticles; and    -   (B) probe comprising a PA molecule or group of PA molecules        bound (through a linker, which can be fixed or detachable) to an        energy modulation agent particle that can produce excitons under        radiative excitation at a suitable wavelength (e.g., X-ray). The        energy modulation agent particle is bound to (or in proximity        of) a metal nanoparticle via a spacer (linker). The spacer is        designed to prevent quenching of the luminescence light emitted        by the energy modulation agent particle excited by X-ray.

FIG. 25 shows yet further embodiments of EPEP probes of the presentinvention:

(A) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is covered with a nanoshell of silica (or otherdielectric material), which is covered by a layer of separatenanostructures (nano islands, nanorods, nanocubes, etc. . . . ) of metal(preferably Au, Ag). The silica layer (or other dielectric material) isdesigned to prevent quenching of the luminescence light emitted by theEEC (also referred to as energy modulation agent) particle excited byX-ray. The metal nanostructures (Au, Ag, etc) are designed to induceplasmons that enhance the X ray excitation that subsequently leads to anincrease in the EEC light emission, ultimately enhancing the efficiencyof photoactivation, i.e. phototherapy. The structure of the nanoparticlecan also be designed such that the plasmonics effect also enhance theenergy modulation agent emission light. These processes are due tostrong coupling between excitons (in the energy modulation agentmaterials and plasmons in the metal nanostructures).

(B) probe comprising a group of PA molecules in a particle bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The PA-containingparticle is covered with a layer of metallic nanostructures (preferablyAu, Ag). The metal nanostructures (Au, Ag, etc) are designed to induceplasmons that enhance the energy modulation agent light emission,ultimately enhancing the efficiency of photoactivation, i.e.phototherapy.

(C) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is covered with a nanoshell of silica (or otherdielectric material), which is covered by a layer of metallicnanostructures (Au, Ag). The silica layer (or other dielectric material)is designed to prevent quenching of the luminescence light emitted bythe energy modulation agent particle excited by X-ray. The metalnanostructures (Au, Ag, etc) are designed to induce plasmons thatenhance the X ray excitation that subsequently leads to an increase inthe energy modulation agent light emission, ultimately enhancing theefficiency of photoactivation, i.e. phototherapy. In addition. thePA-containing particle is covered with a layer of metallicnanostructures (Au, Ag). The metal nanostructures (Au, Ag, etc) aredesigned to induce plasmons that enhance the EEC light emission,ultimately enhancing the efficiency of photoactivation, i.e.phototherapy.

Hybrid EPEP Nano-Superstructures

EPEP probes can also comprise hybrid self-assembled superstructures madeof biological and abiotic nanoscale components, which can offerversatile molecular constructs with a spectrum of unique electronic,surface properties and photospectral properties for use in phototherapy.

Biopolymers and nanoparticles can be integrated in superstructures,which offer unique functionalities because the physical properties ofinorganic nanomaterials and the chemical flexibility/specificity ofpolymers can be used. Noteworthy are complex systems combining two typesof excitations common in nanomaterials, such as excitons and plasmonsleading to coupled excitations. Molecular constructs comprising buildingblocks including metal, semiconductor nanoparticles (NPs), nanorods(NRs) or nanowires (NWs) can produce EPEP probes with an assortment ofphotonic properties and enhancement interactions that are fundamentallyimportant for the field of phototherapy. Some examples of assemblies ofsome NW nanostructures and NPs have been reported in biosensing.Nanoscale superstructures made from CdTe nanowires (NWs) and metalnanoparticles (NPs) are prepared via bioconjugation reactions.Prototypical biomolecules, such as D-biotin and streptavidin pair, wereutilized to connect NPs and NWs in solution. It was found that Au NPsform a dense shell around a CdTe NW. The superstructure demonstratedunusual optical effects related to the long-distance interaction of thesemiconductor and noble metal nanocolloids. The NWNP complex showed5-fold enhancement of luminescence intensity and a blue shift of theemission peak as compared to unconjugated NW. [Jaebeom Lee, † AlexanderO. Govorov, ‡ John Dulka, ‡ and Nicholas A. Kotov*, †, Bioconjugates ofCdTe Nanowires and Au Nanoparticles: Plasmon-Exciton Interactions,Luminescence Enhancement, and Collective Effects, Nano Lett., Vol. 4,No. 12, 2323, 2004].

To the present inventors' knowledge, these advanced concepts have notbeen applied to phototherapy and EPEP probes comprising superstructuresfrom NPs, NRs and NWs are still a new unexplored territory ofphototherapy.

FIG. 25 shows various embodiments of EPEP probes of the presentinvention comprising superstructures of NPs, NWs and NRs.:

(A) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanowire (ornanorod) covered with a nanoshell cylinder of silica (or otherdielectric material). The silica nanoshells cylinder is designed toprevent quenching of the luminescence light emitted by the energymodulation agent particle excited by X-ray. The metal nanoparticle (Au,Ag, etc) is designed to induce plasmons that enhance the X rayexcitation that subsequently leads to an increase in the energymodulation agent light emission, ultimately enhancing the efficiency ofphotoactivation, i.e. phototherapy. The structure of the nanoparticlecan also be designed such that the plasmonics effect and/or theexciton-plasmon coupling (EPC) effect also enhances the energymodulation agent emission light. These processes are due to strongcoupling between excitons (in the energy modulation agent materials andplasmons in the metal nanoparticles; and

(B) probe comprising a PA molecule or group of PA molecules bound(through a linker, which can be fixed or detachable) to an energymodulation agent particle that can produce excitons under radiativeexcitation at a suitable wavelength (e.g., X-ray). The energy modulationagent particle is bound to (or in proximity of) a metal nanoparticlesvia a spacer (linker). The spacer is designed to prevent quenching ofthe luminescence light emitted by the energy modulation agent particleexcited by X-ray. Same effect as above in (A)

FIG. 26 shows another set of embodiments of EPEP probes of the presentinvention comprising superstructures of NPs, NWs and NRs andbioreceptors (antibodies, DNA, surface cell receptors, etc.). The use ofbioreceptors to target tumor cells has been discussed previously abovein relation to PEPST probes. Note that in this embodiment the PAmolecules are attached along the NW axis in order to be excited by theemitting light form the NWs.

FIG. 27 shows another embodiment of EPEP probes of the present inventioncomprising superstructures of NPs linked to multiple NWs.

For some embodiments, by adding metal nanostructures designed tointeract specifically with the excitons in the energy modulation agentsystem, there are significant improvements:

(1) an additional radiative pathway from exciton to photon conversion isintroduced

(2) the metal nanostructures can be designed to amplify (due to theplasmonics effect) the excitation radiation (e.g., X-ray) and/or theemission radiation (e.g, UV or visible) to excite the photo-active (PA)molecule, thereby enhancing the PA effectiveness.

Various metallic nanostructures that can be used in EPEP probeembodiments of the present invention are the same as those illustratedin FIG. 4 for the PEPST probes.

EPEP Probes with Microresonators

In a preferred embodiment the energy modulation agent system can bedesigned to serve also as a microresonator having micron or submicronsize. Lipson et al described a resonant microcavity and, moreparticularly, to a resonant microcavity which produces a stronglight-matter interaction [M. Lipson; L. C. Kimerling; Lionel C, Resonantmicrocavities, U.S. Pat. No. 6,627,923, 2000]. A resonant microcavity,typically, is formed in a substrate, such as silicon, and has dimensionsthat are on the order of microns or fractions of microns. The resonantmicrocavity contains optically-active matter (i.e., luminescentmaterial) and reflectors which confine light in the optically-activematter. The confined light interacts with the optically-active matter toproduce a light-matter interaction. The light-matter interaction in amicrocavity can be characterized as strong or weak. Weak interactions donot alter energy levels in the matter, whereas strong interactions alterenergy levels in the matter. In strong light-matter interactionarrangements, the confined light can be made to resonate with theseenergy level transitions to change properties of the microcavity.

Experimental Methods

Preparation of Nanoparticles (Ag, Au)

There many methods to prepare metal nanoparticles for EPEP or PEPSTprobes. Procedures for preparing gold and silver colloids includeelectroexplosion, electrodeposition, gas phase condensation,electrochemical methods, and solution-phase chemical methods. Althoughthe methodologies for preparing homogeneous-sized spherical colloidalgold populations 2-40 nm in diameter are well known [N. R. Jana, L.Gearheart and C. J Murphy, Seeding growth for size control of 5-40 nmdiameter gold nanoparticles. Langmuir 17 (2001), pp. 6782-6786], andparticles of this size are commercially available. An effective chemicalreduction method for preparing populations of silver particles (withhomogeneous optical scattering properties) or gold particles (withimproved control of size and shape monodispersity) is based on the useof small-diameter uniform-sized gold particles as nucleation centers forthe further growth of silver or gold layers.

A widely used approach involves citrate reduction of a gold salt toproduce 12-20 nm size gold particles with a relatively narrow sizedistribution. The commonly used method for producing smaller goldparticles was developed by Brust et al [Brust, M.; Walker, M.; Bethell,D.; Schiffrin, D. J; Whyman, R. Chem. Commun. 1994, 801]. This method isbased on borohydride reduction of gold salt in the presence of analkanethiol capping agent to produce 1-3 nm particles. Nanoparticlesizes can be controlled between 2 and 5 nm by varying the thiolconcentration, [Hostetler, M. J; Wingate, J. E.; Zhong, C. J.; Harris,J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J; Stokes,J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.;Murray, R. W Langmuir 1998, 14, 17]. Phosphine-stabilized gold clustershave also been produced and subsequently converted to thiol-cappedclusters by ligand exchange in order to improve their stability [Schmid,G.; Pfeil, R.; Boese, R.; Bandrmann, F.; Meyer, S.; Calis, G. H. M.; vander Velden, J. W. A. Chem. Ber. 1981, 114, 3634; Warner, M. G.; Reed, S.M.; Hutchison, J. E. Chem. Mater. 2000, 12, 3316.] andphosphine-stabilized monodispersed gold particles were prepared using asimilar protocol to the Brust method [Weare, W. W.; Reed, S. M.; Warner,M. G.; Hutchison, J. E. J. Am. Chem. Soc. 2000, 122, 12890]. See alsorecent review: Ziyi Zhong, Benoit¹ Male, Keith B.¹ Luong, John H. T.,More Recent Progress in the Preparation of Au Nanostructures,Properties, and Applications, Analytical Letters; 2003, Vol. 36 Issue15, p 3097-3118]

Fabrication of Nanoparticle of Metal Coated with Nanoshells of Dyes

The fabrication of metal nanoparticles coated with nanoshells of dyemolecules can be performed using the method described by Masuhara et al[AKITO MASUHARA_, SATOSHI OHHASHIy, HITOSHI KASAI, SHUJI OKADA,FABRICATION AND OPTICAL PROPERTIES OF NANOCOMPLEXES COMPOSED OF METALNANOPARTICLES AND ORGANIC DYES, Journal of Nonlinear Optical Physics &Materials Vol. 13, Nos. 3 & 4 (2004) 587-592]. Nanocomplexes composed ofAg or Au as a core and3-carboxlymethyl-5-[2-(3-octadecyl-2-benzoselenazolinylidene)ethylidene]rhodanine(MCSe) or copper (II) phthalocyanine (CuPc) as a shell are prepared bythe co-reprecipitation method. In the case of Ag-MCSe nanocomplexes, 0.5mM acetone solution of MCSe are injected into 10 ml of Ag nanoparticlewater dispersion, prepared by the reduction of AgNO₃ using NaBH₄:Au-MCSe nanocomplexes are also fabricated in a similar manner. A waterdispersion of Au nanoparticles was prepared by the reduction of HAuCl₄using sodium citrate. Subsequently, 2 M NH₄OH (50 μl) was added and themixture was thermally treated at 50° C. This amine treatment oftenstimulates the J-aggregate formation of MCSe. 6 Ag—CuPc and Au—CuPcnanocomplexes were also fabricated in the same manner: 1 mM1-methyl-2-pyrrolidinone (NMP) solution of CuPc (200 μl) was injectedinto a water dispersion (10 ml) of Ag or Au nanoparticles.

The present invention treatment may also be used for inducing an autovaccine effect for malignant cells, including those in solid tumors. Tothe extent that any rapidly dividing cells or stem cells may be damagedby a systemic treatment, then it may be preferable to direct thestimulating energy directly toward the tumor, preventing damage to mostnormal, healthy cells or stem cells by avoiding photoactivation orresonant energy transfer of the photoactivatable agent.

Alternatively, a treatment may be applied that slows or pauses mitosis.Such a treatment is capable of slowing the division of rapidly dividinghealthy cells or stem cells during the treatment, without pausingmitosis of cancerous cells. Alternatively, a blocking agent isadministered preferentially to malignant cells prior to administeringthe treatment that slows mitosis.

In one embodiment, an aggressive cell proliferation disorder has a muchhigher rate of mitosis, which leads to selective destruction of adisproportionate share of the malignant cells during even a systemicallyadministered treatment. Stem cells and healthy cells may be spared fromwholesale programmed cell death, even if exposed to photoactivatedagents, provided that such photoactivated agents degenerate from theexcited state to a lower energy state prior to binding, mitosis or othermechanisms for creating damage to the cells of a substantial fraction ofthe healthy stem cells. Thus, an auto-immune response may not beinduced.

Alternatively, a blocking agent may be used that prevents or reducesdamage to stem cells or healthy cells, selectively, which wouldotherwise be impaired. The blocking agent is selected or is administeredsuch that the blocking agent does not impart a similar benefit tomalignant cells, for example.

In one embodiment, stem cells are targeted, specifically, fordestruction with the intention of replacing the stem cells with a donorcell line or previously stored, healthy cells of the patient. In thiscase, no blocking agent is used. Instead, a carrier or photosensitizeris used that specifically targets the stem cells.

Work in the area of photodynamic therapy has shown that the amount ofsinglet oxygen required to cause cell lysis, and thus cell death, is0.32×10⁻³ mol/liter or more, or 109 singlet oxygen molecules/cell ormore. However, in one embodiment of the present invention, it is mostpreferable to avoid production of an amount of singlet oxygen that wouldcause cell lysis, due to its indiscriminate nature of attack, lysingboth target cells and healthy cells. Accordingly, it is most preferredin the present invention that the level of singlet oxygen productioncaused by the initiation energy used or activatable pharmaceutical agentupon activation be less than level needed to cause cell lysis.

In a further embodiment, methods in accordance with the presentinvention may further include adding an additive to alleviate treatmentside-effects. Exemplary additives may include, but are not limited to,antioxidants, adjuvant, or combinations thereof. In one exemplaryembodiment, psoralen is used as the activatable pharmaceutical agent,UV-A is used as the activating energy, and antioxidants are added toreduce the unwanted side-effects of irradiation.

The activatable pharmaceutical agent and derivatives thereof as well asthe energy modulation agent and plasmonics compounds and structures, canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the activatablepharmaceutical agent and a pharmaceutically acceptable carrier. Thepharmaceutical composition also comprises at least one additive having acomplementary therapeutic or diagnostic effect, wherein the additive isone selected from an antioxidant, an adjuvant, or a combination thereof.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions. Modifications can be made to the compound of thepresent invention to affect solubility or clearance of the compound.These molecules may also be synthesized with D-amino acids to increaseresistance to enzymatic degradation. If necessary, the activatablepharmaceutical agent can be co-administered with a solubilizing agent,such as cyclodextran.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, rectal administration, and direct injection into theaffected area, such as direct injection into a tumor. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerin, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates, and agents for the adjustment of tonicity suchas sodium chloride or dextrose. The pH can be adjusted with acids orbases, such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack,kit or dispenser together with instructions for administration.

Methods of administering agents according to the present invention arenot limited to the conventional means such as injection or oralinfusion, but include more advanced and complex forms of energytransfer. For example, genetically engineered cells that carry andexpress energy modulation agents may be used. Cells from the host may betransfected with genetically engineered vectors that expressbioluminescent agents. Transfection may be accomplished via in situ genetherapy techniques such as injection of viral vectors or gene guns, ormay be performed ex vivo by removing a sample of the host's cells andthen returning to the host upon successful transfection.

Such transfected cells may be inserted or otherwise targeted at the sitewhere diseased cells are located. In this embodiment, the initiationenergy source may be a biochemical source as such ATP, in which case theinitiation energy source is considered to be directly implanted in thetransfected cell. Alternatively, a conventional micro-emitter devicecapable of acting as an initiation energy source may be transplanted atthe site of the diseased cells.

It will also be understood that the order of administering the differentagents is not particularly limited. Thus in some embodiments theactivatable pharmaceutical agent may be administered before the energymodulation agent, while in other embodiments the energy modulation agentmay be administered prior to the activatable pharmaceutical agent. Itwill be appreciated that different combinations of ordering may beadvantageously employed depending on factors such as the absorption rateof the agents, the localization and molecular trafficking properties ofthe agents, and other pharmacokinetics or pharmacodynamicsconsiderations.

An advantage of the methods of the present invention is that byspecifically targeting cells affected by a cell proliferation disorder,such as rapidly dividing cells, and triggering a cellular change, suchas apoptosis, in these cells in situ, the immune system of the host maybe stimulated to have an immune response against the diseased cells.Once the host's own immune system is stimulated to have such a response,other diseased cells that are not treated by the activatablepharmaceutical agent may be recognized and be destroyed by the host'sown immune system. Such autovaccine effects may be obtained, forexample, in treatments using psoralen and UV-A.

The present invention methods can be used alone or in combination withother therapies for treatment of cell proliferation disorders.Additionally, the present invention methods can be used, if desired, inconjunction with recent advances in chronomedicine, such as thatdetailed in Giacchetti et al, Journal of Clinical Oncology, Vol 24, No22 (August 1), 2006: pp. 3562-3569. In chronomedicine it has been foundthat cells suffering from certain types of disorders, such as cancer,respond better at certain times of the day than at others. Thus,chronomedicine could be used in conjunction with the present methods inorder to augment the effect of the treatments of the present invention.

Another embodiment of the present invention is a computer-implementedsystem, comprising:

a central processing unit (CPU) having a storage medium on which isprovided:

a database of excitable compounds;

a database of plasmonics-active agents;

a first computation module for identifying and designing an excitablecompound that is capable of activation and of binding with a targetcellular structure or component;

a second computation module predicting the resonance absorption energyof the excitable compound; and

a third computation module predicting the plasmonics-enhancedspectroscopic properties of the plasmonics-active agents,

wherein the system, upon selection of a target cellular structure orcomponent, computes an excitable compound that is capable of activationand of binding with the target structure followed by a computation topredict the resonance absorption energy of the excitable compound and bya computation of the plasmonics-enhanced spectroscopic properties of thephotonics-active agents.

The computer implemented system can further comprise an energyinitiation source connected to the CPU, wherein after computation of theresonance absorption energy of the excitable compound and theplasmonics-enhanced spectroscopic properties of the photonics-activeagents, the system directs the energy initiation source to provide thecomputed resonance absorption energy to the excitable compound. In sucha computer implemented system, the plasmonics active agent is preferablya PEPST probe or EPEP probe.

In another aspect, the present invention further provides systems andkits for practicing the above described methods.

In one embodiment, a system in accordance with the present invention mayinclude: (1) an initiation energy source; (2) one or more energymodulation agents; and (3) one or more activatable pharmaceuticalagents.

In another embodiment, a system in accordance with the present inventionmay include an initiation energy source and one or more activatablepharmaceutical agents.

In preferred embodiments, the initiation energy source may be a linearaccelerator equipped with image guided computer-control capability todeliver a precisely calibrated beam of radiation to a pre-selectedcoordinate. One example of such linear accelerators is the SmartBeam™IMRT (intensity modulated radiation therapy) system from Varian medicalsystems (Varian Medical Systems, Inc., Palo Alto, Calif.).

In other embodiments, endoscopic or laparoscopic devices equipped withappropriate initiation energy emitter may be used as the initiationenergy source. In such systems, the initiation energy may be navigatedand positioned at the pre-selected coordinate to deliver the desiredamount of initiation energy to the site.

In further embodiments, dose calculation and robotic manipulationdevices may also be included in the system.

The reagents and chemicals useful for methods and systems of the presentinvention may be packaged in kits to facilitate application of thepresent invention. In one exemplary embodiment, a kit including apsoralen, and fractionating containers for easy fractionation andisolation of autovaccines is contemplated. A further embodiment of kitwould comprise at least one activatable pharmaceutical agent capable ofcausing a predetermined cellular change, at least one energy modulationagent capable of activating the at least one activatable agent whenenergized, at least one plasmonics agent and containers suitable forstoring the agents in stable form, and preferably further comprisinginstructions for administering the at least one activatablepharmaceutical agent, at least one plasmonics agent and at least oneenergy modulation agent to a subject, and for applying an initiationenergy from an initiation energy source to activate the activatablepharmaceutical agent. The instructions could be in any desired form,including but not limited to, printed on a kit insert, printed on one ormore containers, as well as electronically stored instructions providedon an electronic storage medium, such as a computer readable storagemedium. Also optionally included is a software package on a computerreadable storage medium that permits the user to integrate theinformation and calculate a control dose, to calculate and controlintensity of the irradiation source.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Preparation of Silver Nanoparticles

Silver (or gold) colloids were prepared according to the standardLee-Meisel method: 200 mL of 10⁻³ M AgNO₃ aqueous solution was boiledunder vigorous stirring, then 5 mL of 35-mM sodium citrate solution wereadded and the resulting mixture was kept boiling for 1 h. This procedurewas reported to yield ˜10¹¹ particles/mL of homogenously sized colloidalparticles with a diameter of ˜35-50 nm and an absorption maximum at 390nm. The colloidal solutions were stored at 4° C. and protected from roomlight. Further dilutions of the colloidal solutions were carried outusing distilled water.

Fabrication/Preparation of Metal Nanocaps

One approach has involved the use of nanospheres spin-coated on a solidsupport in order to produce and control the desired roughness. Thenanostructured support is subsequently covered with a layer of silverthat provides the conduction electrons required for the surface plasmonmechanisms. Among the techniques based on solid substrates, the methodsusing simple nanomaterials, such as Teflon or latex nanospheres, appearto be the simplest to prepare. Teflon and latex nanospheres arecommercially available in a wide variety of sizes. The shapes of thesematerials are very regular and their size can be selected for optimalenhancement. These materials comprise isolated dielectric nanospheres(30-nm diameter) coated with silver producing systems ofhalf-nanoshells, referred to as nanocaps.

FIG. 19 shows a scanning electron micrograph (SEM) of 300-nm diameterpolymer nanospheres covered by a 100-nm thick silver nanocaps(half-nanoshell) coating. The nanoparticles can be sonicated to releasethem from the underlying substrate. The effect of the sphere size andmetal layer thickness upon the SERS effect can be easily investigated.The silver coated nanospheres were found to be among the mostplasmonics-active investigated. Gold can also be used instead of silverto coat over nanoparticles comprising PA drug molecules.

Fabrication of Gold Nanoshells

Gold nanoshells have been prepared using the method described by Hirschet al. [Hirsch L R, Stafford R J, Bankson J A, Sershen S R, Price R E,Hazle J D, Halas N J, West J L (2003) Nanoshell-mediated near infraredthermal therapy of tumors under M R Guidance. Proc Natl Acad Sci100:13549-13554] using a mechanism involving nucleation and thensuccessive growth of gold nanoparticles around a silica dielectric core.Gold nanoparticles, the seed, prepared as described above using theFrens method, were used to grow the gold shell. Silica nanoparticles(100 nm) used for the core of the nanoshells were monodispersed insolution of 1% APTES in EtOH. The gold “seed” colloid synthesized usingthe Frens method were grown onto the surface of silica nanoparticles viamolecular linkage of amine groups.

The “seed” covers the aminated silica nanoparticle surface, first as adiscontinuous gold metal layer gradually growing forming a continuousgold shell. Gold nanoparticles used as the “seed” were characterizedusing optical transmission spectroscopy (UV-Vis Spectrophotometer,Beckman Coulter, Fullerton, Calif.) and atomic force microscopy (AtomicForce Microscope, Veeco Instruments, Woodbury, N.Y.) while goldnanoshells were characterized using optical transmission spectroscopyand scanning electron microscopy (Scanning Electron Microscope, HitachiS-4700, Hitachi High Technologies America, Inc. Pleasanton, N.Y.).

Probe for Measurement of Apoptosis with the PDT Drug ALA

A method has been developed using nanosensors that can be used toevaluate the effectiveness of PEPST probes. Although one can useconventional methods (not requiring nanosensors), we describe thenanosensor method previously developed [P. M. Kasili, J M. Song, and TVo-Dinh, “Optical Sensor for the Detection of Caspase-9 Activity in aSingle Cell”, J. Am. Chem. Soc., 126, 2799-2806 (2004)]. The methodcomprises measuring caspases activated by apoptosis induced by thephotoactive drugs. In this experiment, we measure two sets of cells Iand II. Set I is treated with the drug ALA and set II is treated by thedrug ALA conjugated to a PEPST probe described in the previous section.By comparing the results (amount of Caspases detected), one can evaluatethe efficiency of the PEPST-ALA drug compared to ALA alone.

In the classical model of apoptosis, caspases are divided into initiatorcaspases and effector caspases according to their function and theirsequence of activation. Initiator caspases include caspase-8, -9, whileeffector caspases include, caspases-3, -6 and -7. The activation ofcaspases is one of the earliest biomarkers of apoptosis making caspasesan early and ideal target for measuring apoptosis. Apoptosis, orprogrammed cell death, is a mode of cell death characterized by specificmorphological and biochemical features. The results obtained in theseexperiments can be used to evaluate the effectiveness ofphototherapeutic drugs that induce apoptosis (e.g. PDT drugs). Sincecaspases play a central role in the induction of apoptosis,tetrapeptide-based optical nanosensors were used to determine their rolein response to a photodynamic therapy (PDT) agent, 6-aminolevulinic acid(ALA) in the well-characterized human breast carcinoma cell line, MCF-7.MCF-7 cells were exposed to the photosensitizer ALA to explore ALA-PDTinduced apoptosis by monitoring caspase-9 and caspase-7 activity.Caspase-9 and caspase-7 protease activity was assessed in single livingMCF-7 cells with the known caspase-9 and caspase-7 substrates,Leucine-aspartic-histidine-glutamic acid 7-amino-4-methylcoumarin(LEHD-AMC) and aspartic-glutamic acid-valine-aspartic acid7-amino-4-methylcoumarin (DEVD-AMC) respectively, covalently immobilizedto the nanotips of optical nanosensors. Upon the induction of apoptosis,activated target caspases recognize the tetrapeptide sequence andspecifically cleaves it. The recognition of substrate by caspases isimmediately followed by a cleavage reaction yielding the fluorescent AMCwhich can be excited with a Helium-Cadmium (HeCd) laser to generate ameasurable fluorescence signal. By comparing the fluorescence signalgenerated from AMC within cells with activated caspases and from thosewith inactive caspases, we are able to successfully detect caspaseactivity within a single living MCF-7 cell.

Chemicals and Reagents

δ-aminolevulinic acid (ALA), phosphate buffered saline (PBS),hydrochloric acid (HCl), nitric acid (HNO₃),Glycidoxypropyltrimethoxysilane (GOPS), 1,1′-Carbonyldiimidazole (CDI),and anhydrous acetonitrile were purchased from Sigma-Aldrich, St. Louis,Mo. Caspase-9 substrate, LEHD-7-amino-4-methylcoumarin (AMC), Caspase-7substrate, DEVD-7-amino-4-methylcoumarin (AMC), 2× reaction buffer,dithiothreitol (DTT), and dimethylsulfoxide (DMSO) were purchased fromBD Biosciences, Palo Alto. Calif.

Cell Lines

Human breast cancer cell line, MCF-7, was obtained from American TypeCulture Collection (Rockville, Md., USA, Cat-no. HTB22). MCF-7 cellswere grown in Dulbecco's Modified Eagle's Medium ((DMEM) (Mediatech,Inc., Herndon, Va.)) supplemented with 1 mM L-glutamine (Gibco, GrandIsland, N.Y.) and 10% fetal bovine serum (Gibco, Grand Island, N.Y.).Cell culture was established in growth medium (described above) instandard T25 tissue culture flasks (Corning, Corning, N.Y.). The flaskswere incubated in a humidified incubator at 37° C., 5% CO₂ and 86%humidity. Cell growth was monitored daily by microscopic observationuntil a 60-70% state of confluence was achieved. The growth conditionswere chosen so that the cells would be in log phase growth duringphotosensitizer treatment with ALA, but would not be so close toconfluence that a confluent monolayer would form by the termination ofthe chemical exposure. In preparation for experiments, cells wereharvested from the T25 flasks and 0.1 ml (10⁵ cells/ml) aliquots wereseeded into 60 mm tissue culture dishes (Corning Costar Corp., Corning,N.Y.) for overnight attachment. The MCF-7 cells were studied as fourseparate groups with the first group, Group I, being the experimental,exposed to 0.5 mM ALA for 3 h followed by photoactivation([+]ALA[+]PDT). This involved incubating the cells at 37° C. in 5% CO₂for 3 h with 0.5 mM ALA. Following incubation the MCF-7 cells wereexposed to red light from a HeNe laser (λ632.8 nm, <15 mW, Melles Griot,Carlsbad, Calif.) positioned about 5.0 cm above the cells for fiveminutes at a fluence of 5.0 mJ/cm² to photoactivate ALA and subsequentlyinduce apoptosis. The second and third groups, Group II and IIIrespectively, served as the “treated control” and were exposed to 0.5 mMALA for 3 hours without photoactivation ([+]ALA[−]PDT) andphotoactivation without 0.5 mM ALA ([−]ALA[+]PDT]) respectively. Thefourth group, Group IV was the “untreated control,” which receivedneither ALA nor photoactivation ([−]ALA[−]PDT

Experimental Protocol

Preparation of Enzyme Substrate-Based Optical Nanosensors

Briefly, this process involved cutting and polishing plastic clad silica(PCS) fibers with a 600-μm-size core (Fiberguide Industries, Stirling,N.J.). The fibers were pulled to a final tip diameter of 50 nm and thencoated with ˜100 nm of silver metal (99.999% pure) using a thermalevaporation deposition system (Cooke Vacuum Products, South Norwalk,Conn.) achieving a final diameter of 150 nm. The fused silica nanotipswere acid-cleaned (HNO₃) followed by several rinses with distilledwater. Finally, the optical nanofibers were allowed to air dry at roomtemperature in a dust free environment. The nanotips were then silanizedand treated with an organic coupling agent, 10%Glycidoxypropyltrimethoxysilane (GOPS) in distilled water. Thesilanization agent covalently binds to the silica surface of thenanotips modifying the hydroxyl group to a terminus that is compatiblewith the organic cross-linking reagent, 1′1, Carbonyldiimidazole (CDI).The use of CDI for activation introducing an imidazole-terminal groupwas particularly attractive since the protein to be immobilized could beused without chemical modification. Proteins bound using this procedureremained securely immobilized during washing or subsequent manipulationsin immunoassay procedures, as opposed to procedures that use adsorptionto attach proteins. The silanized and activated nanotips for measuringcaspase-9 activity were immersed in a solution containing DMSO, 2×reaction buffer, PBS, and LEHD-AMC, and allowed to incubate for 3 h at37° C., while those for measuring caspase-7 activity were immersed in asolution containing DMSO, 2× reaction buffer, PBS, and DEVD-AMC, andallowed to incubate for 3 h at 37° C.

Measurement System and Procedure

A schematic representation of the experimental setup used in this workis described in a previous work [[P.M. Kasili, J. M. Song, and T.Vo-Dinh, “Optical Sensor for the Detection of Caspase-9 Activity in aSingle Cell”, J. Am. Chem. Soc., 126, 2799-2806 (2004)].The componentsincluded a HeCd laser (Omnichrome, <5 mW laser power) for excitation, anoptical fiber for delivery of excitation light to the opticalnanosensor, a Nikon Diaphot 300 inverted fluorescence microscope (Nikon,Inc., Melville, N.Y.), a photon counting photomultiplier tube (PMT) anda PC for data acquisition and processing. This experimental set-up, usedto probe single cells, was adapted for this purpose from a standardmicromanipulation and microinjection apparatus. The Nikon Diaphot 300inverted microscope was equipped with a Diaphot 300/Diaphot 200Incubator to maintain the cell cultures at 37° C. on the microscopestage, during these experiments. The micromanipulation equipmentconsisted of MN-2 (Narishige Co. Ltd., Tokyo, Japan) Narishigethree-dimensional manipulators for coarse adjustment, and NarishigeMMW-23 three-dimensional hydraulic micromanipulators for fineadjustments. The optical nanosensor was mounted on a micropipette holder(World Precision Instruments, Inc., Sarasota, Fla.). The 325 nm laserline of a HeCd laser was focused onto a 600-μm-delivery fiber that isterminated with a subminiature A (SMA) connector. The enzymesubstrate-based optical nanosensor was coupled to the delivery fiberthrough the SMA connector and secured to the Nikon inverted microscopewith micromanipulators. To record the fluorescence generated by AMCmolecules at the nanotips, a Hamamatsu PMT detector assembly (HC125-2)was mounted in the front port of the Diaphot 300 microscope. Thefluorescence emitted by AMC from the measurement made using single livecells was collected by the microscope objective and passed through a330-380 nm filter set and then focused onto a PMT for detection. Theoutput from the PMT was recorded using a universal counter interfaced toa personal computer (PC) for data treatment and processing.

In Vitro Determination of Caspase Activity

After incubation using the following treatment groups, group(I)-[+]ALA[+]PDT, group II-[+]ALA[−]PDT, group III-[−]ALA[+]PDT, andgroup IV-[−]ALA[−]PDT, MCF-7 cells were washed with PBS solution, pH7.4, and then resuspended in lysis buffer (100 mM HEPES, pH 7.4, 10%sucrose, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CHAPS), 1 mM EDTA, 10 mM dithiothreitol (DTT), 1 mMphenylmethylsulphonyl fluoride (PMSF), 10 mg/ml pepstatin, 10 mg/mlleupeptin) and left on ice for 45 minutes. The cells were thenrepeatedly passed through a syringe with a 25-gauge needle until most ofthe cell membrane was disrupted, and centrifuged at 1500 RPM for 10 min.Activity of caspases was measured using the fluorogenic substratepeptides; LEHD-AMC for caspase-9 and DEVD-AMC for caspase-7. The releaseof AMC was measured after incubating optical nanosensors in picofugetubes containing the cell lysates from the various treatment groups andusing a HeCd laser (excitation 325 nm) to excite AMC. Caspase activitywas expressed as fluorescence intensity of AMC as a function ofequivalent nanomoles of LEHD-AMC and DEVD-AMC respectively.

The results of the in vitro measurement of caspase-9 and caspase-7activity were plotted. The curves for each fluorescent measurement ofAMC were plotted for each as a function of AMC concentration. Caspase-9activity was determined by incubation of optical nanosensors with thesubstrate LEHD-7-amino-4-methylcoumarin (AMC) in cell lysate (˜10⁵cells) obtained from the following treatment groups; group I, II, I andIV, described earlier in the article. The release of AMC was measuredafter excitation using HeCd laser (325 nm) and collecting thefluorescence signal using a 380 nm longpass filter. The peak emissionwavelength of AMC is about 440 nm. Likewise, Caspase-7 activity wasdetermined by incubation in cell lysate (˜10⁵ cells) obtained from thefollowing treatment groups I, II, III, and IV. The release of AMC wasmeasured after excitation using a HeCd laser (325 nm) and collecting thefluorescence signal using a 380 nm longpass filter.

In this experiment, we measure two sets of cells I and II: (1) Set I istreated with the drug ALA and (2) set II is treated by the drug ALAconjugated to a PEPST probe described in the previous section. Bycomparing the results (amount of caspase detected), one can evaluate theefficiency of the PEPST-ALA drug compared to ALA alone.

Additional modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

1. A method for treating a cell proliferation disorder in a subject,comprising: (1) administering to the subject at least one activatablepharmaceutical agent that is capable of effecting a predeterminedcellular change when activated and at least one plasmonics-active agent,and (3) applying an initiation energy from an initiation energy sourceto the subject, wherein the plasmonics-active agent enhances or modifiesthe applied initiation energy, such that the enhanced or modifiedinitiation energy activates the activatable pharmaceutical agent insitu, thus causing the predetermined cellular change to occur, whereinsaid predetermined cellular change treats the cell proliferation relateddisorder.
 2. The method of claim 1, wherein the initiation energy sourceis selected from UV radiation, visible light, infrared radiation,x-rays, gamma rays, electron beams, phosphorescent compounds,chemiluminescent compounds, bioluminescent compounds, and light emittingenzymes.
 3. The method of claim 1, wherein the initiation energy sourceis x-rays, gamma rays, an electron beam, microwaves or radio waves. 4.The method of claim 1, wherein the initiation energy is capable ofpenetrating completely through the subject.
 5. The method of claim 1,wherein the cell proliferation disorder is at least one member selectedfrom the group consisting of cancer, bacterial infection, viralinfection, immune rejection response, autoimmune disorders, aplasticconditions, and combinations thereof.
 6. The method of claim 1, whereinthe at least one activatable pharmaceutical agent is a photoactivatableagent.
 7. The method of claim 1, wherein the at least one activatablepharmaceutical agent is selected from psoralens, pyrenecholesteryloleate, acridine, porphyrin, fluorescein, rhodamine,16-diazorcortisone, ethidium, transition metal complexes of bleomycin,transition metal complexes of deglycobleomycin organoplatinum complexes,alloxazines, vitamin Ks, vitamin L, vitamin metabolites, vitaminprecursors, naphthoquinones, naphthalenes, naphthols and derivativesthereof having planar molecular conformations, porphorinporphyrins, dyesand phenothiazine derivatives, coumarins, quinolones, quinones, andanthroquinones.
 8. The method of claim 6, wherein the at least oneactivatable pharmaceutical agent is a psoralen, a coumarin, a porphyrinor a derivative thereof.
 9. The method of claim 6, wherein the at leastone activatable pharmaceutical agent is 8-MOP or AMT.
 10. The method ofclaim 1, wherein the at least one activatable pharmaceutical agent isone selected from 7,8-dimethyl-10-ribityl, isoalloxazine,7,8,10-trimethylisoalloxazine, 7,8-dimethylalloxazine,isoalloxazine-adenine dinucleotide, alloxazine mononucleotide, aluminum(III) phthalocyanine tetrasulonate, hematophorphyrin, andphthadocyanine.
 11. The method of claim 1, wherein the at least oneactivatable pharmaceutical agent is coupled to a carrier that is capableof binding to a receptor site.
 12. The method of claim 11, wherein thecarrier is one selected from insulin, interleukin, thymopoietin ortransferrin.
 13. The method of claim 11, wherein the at least oneactivatable pharmaceutical agent is coupled to the carrier by a covalentbond.
 14. The method of claim 11, wherein the at least one activatablepharmaceutical agent is coupled to the carrier by non-covalent bond. 15.The method of claim 11, wherein the receptor site is one selected fromnucleic acids of nucleated cells, antigenic sites on nucleated cells, orepitopes.
 16. The method of claim 1, wherein the at least oneactivatable pharmaceutical agent has affinity for a target cell.
 17. Themethod of claim 1, wherein the at least one activatable pharmaceuticalagent is capable of being preferentially absorbed by a target cell. 18.The method of claim 1, wherein the predetermined cellular change isapoptosis in a target cell.
 19. The method of claim 1, wherein the atleast one activated pharmaceutical agent causes an auto-vaccine effectin the subject that reacts with a target cell.
 20. The method of claim19, wherein the auto-vaccine effect is generated in a joint or lymphnode.
 21. The method of claim 1, wherein the at least one activatablepharmaceutical agent is a DNA intercalator or a halogenated derivativethereof.
 22. The method of claim 1, further comprising, prior to saidapplying the initiation energy, administering to the subject at leastone energy modulation agent capable of converting an energy exciting themodulation agent to an energy that activates the at least oneactivatable pharmaceutical agent.
 23. The method of claim 22, whereinthe plasmonic-active agent enhances or modifies the initiation energy tothe energy exciting the energy modulation agent that converts theexcitation energy into the energy that activates the at least oneactivatable pharmaceutical agent.
 24. The method of claim 22, whereinthe plasmonic-active agent enhances or modifies an energy emitted by themodulation agent, to the energy that activates the at least oneactivatable pharmaceutical agent.
 25. The method of claim 22, whereinthe plasmonic-active agent enhances or modifies (a) the initiationenergy to the energy exciting the energy modulation agent that convertsthe excitation energy into the energy that activates the at least oneactivatable pharmaceutical agent, and (b) an energy emitted by themodulation agent, to the energy that activates the at least oneactivatable pharmaceutical agent.
 26. The method of claim 22, whereinsaid at least one energy modulation agent is a single energy modulationagent, and is coupled to said at least one activatable pharmaceuticalagent.
 27. The method of claim 22, wherein a plurality of the energymodulation agents is administered, and wherein the initiation energy, orthe plasmonics-enhanced or modified energy, is converted, through acascade energy transfer between the plurality of the energy modulationagents, to the energy that activates the at least one activatablepharmaceutical agent.
 28. The method of claim 1, wherein the at leastone activatable pharmaceutical agent comprises an active agent containedwithin a photocage, wherein upon exposure to the plasmonics-enhanced ormodified energy, the photocage disassociates from the active agent,rendering the active agent available.
 29. The method of claim 22,wherein the at least one activatable pharmaceutical agent comprises anactive agent contained within a photocage, wherein upon exposure to areemitted energy by the modulation agent as the activation energy of theat least one activatable pharmaceutical agent, the photocagedisassociates from the active agent, rendering the active agentavailable.
 30. The method of claim 24, wherein the at least oneactivatable pharmaceutical agent comprises an active agent containedwithin a photocage, wherein upon exposure to the plasmonics-enhanced ormodified energy emitted by the modulation agent as the activation energyof the at least one activatable pharmaceutical agent, the photocagedisassociates from the active agent, rendering the active agentavailable.
 31. The method of claim 1, wherein said predeterminedcellular change treats the cell proliferation disorder by causing anincrease or decrease in cell proliferation rate of a target cell. 32.The method of claim 1, wherein the plasmonic-active agent enhances theapplied initiation energy from 10⁶ to 10¹⁵ fold.
 33. The method of claim1, wherein the plasmonics-active agent is a plasmonics enhancedphotospectral therapy (PEPST) probe comprising: a) at least one metalnanoparticle, b) at least one activatable pharmaceutical agent, and c)optionally, at least one energy modulation agent.
 34. The method ofclaim 33, wherein at least two of components a)-c) are coupled one toanother.
 35. The method of claim 33, wherein the plasmonics-active agentcomprises a) and b).
 36. The method of claim 33, wherein theplasmonics-active agent comprises a), b), and c).
 37. The method ofclaim 33, wherein the at least one metal nanoparticle is bound to the atleast one energy modulation agent via a linker.
 38. The method of claim33, wherein the energy modulation agent is at least one selected fromthe group consisting of metals, quantum dots, semiconductor materials,scintillation and phosphor materials, materials that exhibit X-rayexcited luminescence (XEOL), organic solids, metal complexes, inorganicsolids, crystals, rare earth materials (lanthanides), polymers,scintillators, phosphor materials, and materials that exhibit excitonicproperties.
 39. The method of claim 1, wherein the plasmonics-activeagent is a PEPST probe having a multi plasmonics resonance mode.
 40. Themethod of claim 1, wherein the plasmonics-active agent is a PEPST probecomprising plasmonics-active metal nanostructures.
 41. The method ofclaim 40, wherein the metal nanostructure is at least one selected fromthe group consisting of a nanosphere, nanorod, nanocube, nanopyramid,nanoshell, nano shell cylinder, and a nano shell or a multi-layer nanoshell.
 42. The method of claim 1, wherein the plasmonics-active agent isa PEPST probe comprising multiple structures for different plasmonicsactivation regimes.
 43. The method of claim 42, wherein said regime isat least one selected from the group consisting of NIR and X ray. 44.The method of claim 1, wherein the plasmonics-active agent is a PEPSTprobe comprising at least one metal nanosystem coupled with theactivatable pharmaceutical agent, or with an energy modulation agentcoupled with the activatable pharmaceutical agent.
 45. The method ofclaim 44, wherein the nanosystem is bound to the activatablepharmaceutical agent or the energy modulation agent by a bond selectedfrom the group consisting of a chemical bond, a biochemical bond, a DNAbond, and an antigen-antibody bond.
 46. The method of claim 44, whereinthe bond is a photo-liable bond.
 47. The method of claim 44, wherein theactivatable pharmaceutical agent is released from the nanosystem insidea cell by photon radiation or ultrasound.
 48. The method of claim 1,wherein the plasmonics-active agent is a PEPST probe comprising at leastone metal nanostructure selected from the group consisting of a metalnanoparticle, a dielectric nanoparticle core covered with a metalnanocap, a spherical metal nanoshell covering a dielectric spheroidcore, a oblate metal nanoshell covering a dielectric spheroid core, ametal nanoparticle core covered with a dielectric nanoshell, a metalnanoshell with a protective coating layer, multi layer metal nanoshellscovering a dielectric spheroid core, multi-nanoparticle structures, ametal nanocube and a nanotriangle/nanoprism, and a metal cylinder. 49.The method of claim 1, wherein the plasmonics-active agent is a PEPSTprobe comprising a combination of metal nanoparticles, covered by alayer of a dielectric material comprising the activatable pharmaceuticalagent, with an energy modulation agent which is bound to the activatablepharmaceutical agent.
 50. The method of claim 1, wherein theplasmonics-active agent is a PEPST probe, which enhances a XEOL lightemitted by a modulation agent irradiated by an X ray, wherein theenhanced XEOL light activates the activatable pharmaceutical agent. 51.The method of claim 1, wherein the plasmonics-active agent is a PEPSTprobe and which is irradiated by X ray to excite the activatablepharmaceutical agent or an energy modulation agent coupled with theactivatable pharmaceutical agent.
 52. The method of claim 1, wherein theplasmonics-active agent is a PEPST probe comprising plasmonics-activemetal nanostructures, said PEPST probe is irradiated by X ray to excitea surface plasmon in nanoparticles or subnanoparticle of a metal. 53.The method of claim 52, wherein the nanoparticles or subnanoparticle ofthe metal are gold or silver nanoparticles or subnanoparticle.
 54. Themethod of claim 52, wherein the metal nanostructure is at least oneselected from the group consisting of a nano sphere, nanorod, nanocube,nanopyramid, nano shell, and a nanoshell or a multi-layer nanoshell. 55.The method of claim 1, wherein the plasmonics-active agent is a PEPSTprobe comprising the activatable pharmaceutical agent or an energymodulation agent coupled with the activatable pharmaceutical agent andfurther comprising at least one bioreceptor.
 56. The method of claim 55,wherein the bioreceptor is at least one selected from the groupconsisting an antibody/antigen, an enzyme, a nucleic acid/DNA, acellular structure/cell, and a biomimetic.
 57. The method of claim 1,comprising delivering to a target the activatable pharmaceutical agentusing a target delivery system comprising the plasmonics-active agentcomprising the activatable pharmaceutical agent and, optionally, anenergy modulation agent, wherein the plasmonics-active agent is a PEPSTprobe.
 58. The method of claim 1, wherein the plasmonics-active agent isa PEPST probe possessing plasmonics photospectral properties,biocompatibility, an improved drug payload delivery and passivetargeting of metal nanoparticles.
 59. The method of claim 33, wherein atleast one biomolecule is immobilized on the metal nanoparticles.
 60. Themethod of claim 59, wherein the biomolecule is the activatablepharmaceutical agent, the modulation agent, a drug, a protein, anenzyme, an antibody, DNA, or RNA.
 61. The method of claim 33, whereinthe metal nanoparticles are gold or silver nanoparticles.
 62. The methodof claim 33, wherein the metal nanoparticles enhance or modify an X rayenergy applied to the subject to excite the energy modulation agentwhich converts the enhanced or modified X ray energy to an energy thatactivates the at least one activatable pharmaceutical agent directly orvia a cascade energy transfer between the plurality of the energymodulation agents.
 63. The method of claim 33, wherein the metalnanoparticles enhance or modify an energy emitted by the modulationagent which converts the initiation energy to the emitted energy, saidenhanced or modified emitted energy activates the at least oneactivatable pharmaceutical agent.
 64. The method of claim 33, whereinthe plasmonics-active agent is a PEPST probe comprising a chain of metalparticles having the same or different size and coupled to one another,wherein the chain of the metal particles exhibits dual or multiplasmonics resonance modes.
 65. The method of claim 64, wherein thechain of particles is used for providing a plasmonics enhancement of theinitiation energy and/or an energy emitted by the modulation agent. 66.The method of claim 1, wherein the activatable pharmaceutical agent isdelivered to a target and is released from the plasmonics-active agentby photon radiation or ultrasound, and wherein the plasmonics-activeagent is a PEPST probe.
 67. The method of claim 22, wherein theactivatable pharmaceutical agent is delivered to a cell and is releasedfrom the energy modulation agent couples with the plasmonics-activeagent by photon radiation or ultrasound, and wherein theplasmonics-active agent is a PEPST probe.
 68. The method of claim 66,wherein the activatable pharmaceutical agent is released from theplasmonics-active agent comprising an antibody or antigen system coupledwith the activatable pharmaceutical agent.
 69. The method of claim 1,comprising delivering the activatable pharmaceutical agent to a targetusing liposomes.
 70. The method of claim 1, wherein theplasmonics-active agent is a PEPST probe comprising an energy modulationagent and the activatable pharmaceutical agent.
 71. The method of claim1, wherein the plasmonics-active agent is a PEPST probe comprising theactivatable pharmaceutical agent which is encapsulated.
 72. The methodof claim 70, wherein the activatable pharmaceutical agent or theactivatable pharmaceutical agent and a modulation agent are encapsulatedin a capsule comprising an ferritin and/or apoferritin compound.
 73. Themethod of claim 1, which uses ultrasound for a release of theactivatable pharmaceutical agent and photonic excitation of theactivatable pharmaceutical agent or an energy modulation agent coupledwith the activatable pharmaceutical agent, wherein the plasmonics-activeagent is a PEPST probe, and the activatable pharmaceutical agent isreleased from the PEPST.
 74. The method of claim 1, the method furthercomprising using a drug delivery, tumor targeting, and/or drug releasingsystem for delivering and releasing the plasmonics-active agent and/orthe activatable pharmaceutical agent, wherein the plasmonics-activeagent is a PEPST probe.
 75. The method of claim 1, wherein theplasmonics-active agent is a PEPST probe, wherein components of thePEPST probe are bound using conjugates, metals binding to organic andinorganic compounds, and biomolecules.
 76. The method of claim 1,wherein the plasmonics-active agent comprises a plasmonic-active metalnanoparticle bound to an excitation energy converter (EEC) materialwhich is bound to the activatable pharmaceutical agent, wherein the EECmaterial for exciton induced phototherapy (EIP) probes is optimizedbased on exciton properties.
 77. The method of claim 1, wherein theplasmonics-active agent comprises an excitation energy converter (EEC)material, said method tunes an emission of the EEC material in excitoninduced phototherapy (EIP) probes to a wavelength capable of exiting theactivatable pharmaceutical agent using specific materials with specificexciton properties, wherein said EEC material can produce excitons undera radiative excitation.
 78. The method of claim 77, wherein the EECmaterial is a modulation agent, said EEC material is bound via a linkerto the activatable pharmaceutical agent.
 79. The method of claim 77,wherein the EEC material is a modulation agent, and the activatablepharmaceutical agent is embedded in a shell around said EEC material.80. The method of claim 77, wherein the EEC material has structuraldefects that serve as traps for the excitation.
 81. The method of claim77, wherein the EEC material comprises impurities or dopant moleculesthat serve as traps for the excitation.
 82. The method of claim 77,wherein the initiation energy is X ray which is transformed to UV orvisible photons by the EEC material.
 83. The method of claim 1, whereinthe plasmonics-active agent is an exciton-plasmon enhanced phototherapy(EPEP) probe which comprises at least one plasmonics-active metalnanostructure, at least one exciton-generating energy modulation agentmaterial, and the at least one activatable pharmaceutical agent, whereinsaid metal nanostructure and said material produce exciton-plasmon (EPC)coupling.
 84. The method of claim 83, wherein the plasmonics-activemetal nanostructure and the exciton-generating energy modulation agentmaterial are coupled via a spacer.
 85. The method of claim 83 or 84,wherein exciton-generating energy modulation agent material is coupledwith the activatable pharmaceutical agent via a linker.
 86. The methodof claim 83 or 84, wherein the activatable pharmaceutical agent and theplasmonics-active metal nanostructure are coupled via a linker.
 87. Themethod of claim 83, wherein the EPEP probe comprises a bioreceptor. 88.The method of claim 87, wherein the bioreceptor is bound to theplasmonics-active metal nanostructure or to the plasmonics-active metalnanostructure covered with a nanoshell or a nanoshell cylinder made of adielectric material.
 89. The method of claim 83 or 84, wherein theplasmonics-active metal nanostructure is covered with a nanoshell madeof a dielectric material.
 90. The method of claim 83 or 84, wherein theplasmonics-active metal nanostructure is a metal nano sphere, a nanowireor a nanorod covered with a nano shell or a nano shell cylinder of adielectric material.
 91. The method of claim 83, wherein the EPEP probecomprises multiple metal nanowires coupled with the exciton-generatingenergy modulation agent material with or without spacers which can bethe same or different, said exciton-generating energy modulation agentmaterial is bound to the activatable pharmaceutical agent via a linker.92. The method of claim 83, wherein the exciton-generating energymodulation agent material is a microresonator.
 93. The method of claim83, wherein the exciton-generating energy modulation agent material isat least one selected from the group consisting of metals, quantum dots,semiconductor materials, scintillation and phosphor materials, materialsthat exhibit X-ray excited luminescence (XEOL), organic solids, metalcomplexes, inorganic solids, crystals, rare earth materials(lanthanides), polymers, scintillators, phosphor materials, andmaterials that exhibit excitonic properties.
 94. The method of claim 83,wherein the metal nanostructure is at least one selected from the groupconsisting of a nanosphere, nanorod, nanocube, nanopyramid, nanoshell,nanoshell cylinder, and multi-layer nanoshells.
 95. The method of claim83, wherein the EPEP probe comprises multiple structures for differentplasmonics activation regimes.
 96. The method of claim 95, wherein saidregime is at least one selected from the group consisting of NIR and Xray.
 97. The method of claim 83, wherein the EPEP probe comprises atleast one metal nanosystem coupled with the activatable pharmaceuticalagent, or with an energy modulation agent coupled with the activatablepharmaceutical agent.
 98. The method of claim 97, wherein the nanosystemis bound to the activatable pharmaceutical agent or the energymodulation agent by a bond selected from the group consisting of achemical bond, a biochemical bond, a DNA bond, and an antigen-antibodybond.
 99. The method of claim 98, wherein the bond is a photo-liablebond.
 100. The method of claim 98, wherein the activatablepharmaceutical agent is released from the nanosystem inside a cell byphoton radiation or ultrasound.
 101. The method of claim 83, wherein theEPEP probe comprises at least one metal nanostructure selected from thegroup consisting of a metal nanoparticle, a dielectric nanoparticle corecovered with a metal nanocap, a spherical metal nanoshell covering adielectric spheroid core, a oblate metal nanoshell covering a dielectricspheroid core, a metal nanoparticle core covered with a dielectricnanoshell, a metal nanoshell with a protective coating layer, multilayer metal nanoshells covering a dielectric spheroid core,multi-nanoparticle structures, a metal nanocube and ananotriangle/nanoprism, and a metal cylinder.
 102. The method of claim83, wherein the EPEP probe comprises a combination of metalnanoparticles with an energy modulation agent which is bound to theactivatable pharmaceutical agent, wherein the metal nanoparticles arecovered by a layer of a dielectric material comprising the activatablepharmaceutical agent.
 103. The method of claim 87, wherein the EPEPprobe enhances a XEOL light emitted by the exciton-generating energymodulation agent material irradiated by an X ray, wherein the enhancedXEOL light activates the activatable pharmaceutical agent.
 104. Themethod of claim 83, wherein the EPEP probe is irradiated by an X ray toexcite the activatable pharmaceutical agent or an energy modulationagent coupled with the activatable pharmaceutical agent.
 105. The methodof claim 83, wherein the EPEP probe comprising the plasmonics-activemetal nanostructures is irradiated by an X ray to excite a surfaceplasmon in nanoparticles or subnanoparticle of a metal.
 106. The methodof claim 105, wherein the nanoparticles or subnanoparticle of the metalare gold or silver nanoparticles or subnanoparticle.
 107. The method ofclaim 105, wherein the metal nanostructure is at least one selected fromthe group consisting of a nanosphere, nanorod, nanocube, nanopyramid,nanoshell, and multi-layer nanoshells.
 108. The method of claim 87,wherein the bioreceptor is at least one selected from the groupconsisting an antibody/antigen, an enzyme, a nucleic acid/DNA, acellular structure/cell, and a biomimetic.
 109. The method of claim 83,comprising delivering to a target the activatable pharmaceutical agentusing a target delivery system comprising the plasmonics-active metalnanostructure comprising the activatable pharmaceutical agent.
 110. Themethod of claim 83, wherein the EPEP probe possesses plasmonicsphotospectral properties, biocompatibility, an improved drug payloaddelivery and passive targeting of metal nanoparticles.
 111. The methodof claim 83, wherein at least one biomolecule is immobilized on themetal nanostructure.
 112. The method of claim 107, wherein thebiomolecule is at least one selected from the group consisting of theactivatable pharmaceutical agent, the modulation agent, a drug, aprotein, an enzyme, an antibody, and a nucleic acid.
 113. The method ofclaim 83, wherein the metal nanostructure is a gold or silvernanoparticle.
 114. The method of claim 83, wherein the metalnanostructure enhances or modifies an X ray energy applied to thesubject to excite the exciton-generating energy modulation agent whichconverts the enhanced or modified X ray energy to an energy thatactivates the at least one activatable pharmaceutical agent directly orvia a cascade energy transfer between the plurality of the energymodulation agents.
 115. The method of claim 83, wherein the metalnanoparticle enhances or modifies an energy emitted by theexciton-generating energy modulation agent which converts the initiationenergy to the emitted energy, said enhanced or modified emitted energyactivates the at least one activatable pharmaceutical agent.
 116. Themethod of claim 83, wherein the metal nanostructure enhances ormodulates (i) an X ray energy applied to the subject to excite theexciton-generating energy modulation agent material which converts theenhanced or modulated X ray energy to an energy that activates the atleast one activatable pharmaceutical agent directly or via a cascadeenergy transfer between the plurality of the energy modulation agents,and (ii) an energy emitted by the exciton-generating energy modulationagent material which converts the enhanced or modulated energy (i) to anenergy that activates the at least one activatable pharmaceutical agent.117. The method of claim 83, wherein (a) the exciton-generating energymodulation agent, (b) the activatable pharmaceutical agent, or (c) theexciton-generating energy modulation agent and the activatablepharmaceutical agent, are covered with a layer comprising theplasmonics-active metal nanostructure.
 118. The method of claim 83,wherein the EPEP probe comprises a chain of metal particles having thesame of different size and coupled to one another, wherein the chain ofthe metal particles exhibit dual or multi plasmonics resonance modes.119. The method of claim 118, wherein the chain of the particles is usedfor providing a plasmonics enhancement of the initiation energy and/oran energy emitted by the exciton-generating energy modulation agentmaterial.
 120. The method of claim 83, wherein the activatablepharmaceutical agent is delivered to a target and is released from theplasmonics-active metal nanostructure by photon radiation or ultrasound.121. The method of claim 120, wherein the activatable pharmaceuticalagent is released from the plasmonics-active metal nanostructurecomprising an antibody or antigen system coupled with the activatablepharmaceutical agent.
 122. The method of claim 83, comprising deliveringthe activatable pharmaceutical agent to a target using liposomes. 123.The method of claim 83, wherein the EPEP probe comprises the activatablepharmaceutical agent which is encapsulated.
 124. The method of claim123, wherein the activatable pharmaceutical agent or the activatablepharmaceutical agent and the exciton-generating energy modulation agentmaterial are encapsulated in a capsule comprising an ferritin and/orapoferritin compound.
 125. The method of claim 83, which uses ultrasoundfor a release of the activatable pharmaceutical agent and photonicexcitation of the activatable pharmaceutical agent or theexciton-generating energy modulation agent material coupled with theactivatable pharmaceutical agent for the activation of the activatablepharmaceutical agent.
 126. The method of claim 1, the method furthercomprising using a drug delivery, tumor targeting, and/or drug releasingsystem for delivering and releasing the plasmonics-active agent and theactivatable pharmaceutical agent, wherein the plasmonics-active agent isa EPEP probe.
 127. The method of claim 1, wherein the plasmonics-activeagent is a EPEP probe, and components of the EPEP probe are bound usingconjugates, metals binding to organic and inorganic compounds, andbiomolecules.
 128. The method of claim 1, wherein the plasmonics agentis a EPEP probe possessing EPIP properties, biocompatibility, animproved drug payload delivery and passive targeting of metalnanoparticles.
 129. The method of claim 1, wherein said condition,disorder, or disease is mediated by abnormal cellular proliferation andsaid predetermined change ameliorates the abnormal cellularproliferation.
 130. The method of claim 129, wherein said abnormalcellular proliferation is higher than that of cells from a subject nothaving said condition, disorder or disease.
 131. The method of claim129, wherein said abnormal cellular proliferation is lower than that ofcells from a subject not having said condition, disorder or disease.132. The method of claim 1, wherein said condition, disorder, or diseaseis not significantly mediated by abnormal cellular proliferation andsaid predetermined change does not substantially affect cellularproliferation.
 133. A method for treating a cell proliferation disorderin a subject, comprising: (1) administering to the subject at least oneenergy modulation agent, at least one activatable pharmaceutical agentthat is capable of effecting a predetermined cellular change whenactivated, and at least one plasmonics-active agent, and (2) applying aninitiation energy from an initiation energy source to the subject,wherein (A) the energy modulation agent upgrades or downgrades theapplied initiation energy, and wherein the plasmonics-active agentenhances or modifies the upgraded or downgraded energy, such that theenhanced or modified upgraded or downgraded energy activates theactivatable pharmaceutical agent in situ, and/or (B) theplasmonics-active agent enhances or modifies the applied initiationenergy, such that the enhanced or modified initiation energy excites themodulation agent which upgrades or downgrades the enhanced or modifiedinitiation energy to an energy that activated the activatablepharmaceutical agent in situ, thus causing the predetermined cellularchange to occur, wherein said predetermined cellular change treats thecell proliferation related disorder.
 134. The method of claim 133,wherein said predetermined cellular change treats the cell proliferationdisorder by causing an increase or decrease in cell proliferation rateof a target cell.
 135. The method of claim 133, wherein the initiationenergy source is x-rays, gamma rays, an electron beam, microwaves orradio waves, wherein the modulation agent upgrades energy.
 136. Themethod of claim 133, wherein the initiation energy source is selectedfrom the group consisting of UV radiation, visible light, infraredradiation, x-rays, gamma rays, electron beams, phosphorescent compounds,chemiluminescent compounds, bioluminescent compounds, and light emittingenzymes.
 137. The method of claim 133, wherein the initiation energysource is a source of lower energy than UV-A, visible energy, and IR orNIR energy, and said at least one energy modulation agent converts theinitiation energy to UV-A, visible energy, IR, or NIR, wherein themodulation agent upgrades energy.
 138. The method of claim 133, whereinthe initiation energy source is a source of higher energy than UV-A orvisible energy and said at least one energy modulation agent convertsthe initiation energy into UV-A or visible energy, wherein themodulation agent downgrades energy.
 139. The method of claim 133,wherein the initiation energy is an IR energy, and the energy activatingthe activatable agent is not UVA or visible light energy.
 140. Themethod of claim 133, wherein the at least one energy modulation agent isone or more selected from the group consisting of a biocompatiblefluorescing metal nanoparticle, fluorescing dye molecule, goldnanoparticle, a water soluble quantum dot encapsulated by polyamidoaminedendrimers, a luciferase, a biocompatible phosphorescent molecule, acombined electromagnetic energy harvester molecule, and a lanthanidechelate capable of intense luminescence.
 141. The method of claim 133,wherein the initiation energy is applied via a thin fiber optic. 142.The method of claim 133, wherein the cell proliferation disorder is atleast one member selected from the group consisting of cancer, bacterialinfection, viral infection, immune rejection response, autoimmunedisorders, aplastic conditions, and combinations thereof.
 143. Themethod of claim 133, wherein the at least one activatable pharmaceuticalagent is a photoactivatable agent.
 144. The method of claim 133, whereinthe at least one activatable pharmaceutical agent is selected frompsoralens, pyrene cholesteryloleate, acridine, porphyrin, fluorescein,rhodamine, 16-diazorcortisone, ethidium, transition metal complexes ofbleomycin, transition metal complexes of deglycobleomycin organoplatinumcomplexes, alloxazines, vitamin Ks, vitamin L, vitamin metabolites,vitamin precursors, naphthoquinones, naphthalenes, naphthols andderivatives thereof having planar molecular conformations,porphorinporphyrins, dyes and phenothiazine derivatives, coumarins,quinolones, quinones, and anthroquinones.
 145. The method of claim 144,wherein the at least one activatable pharmaceutical agent is a psoralen,a coumarin, a porphyrin, or a derivative thereof.
 146. The method ofclaim 143, wherein the at least one activatable pharmaceutical agent is8-MOP or AMT.
 147. The method of claim 133, wherein the at least oneactivatable pharmaceutical agent is one selected from7,8-dimethyl-10-ribityl, isoalloxazine, 7,8,10-trimethylisoalloxazine,7,8-dimethylalloxazine, isoalloxazine-adenine dinucleotide, alloxazinemononucleotide, aluminum (III) phthalocyanine tetrasulonate,hematophorphyrin, and phthadocyanine.
 148. The method of claim 133,wherein the at least one activatable pharmaceutical agent is coupled toa carrier that is capable of binding to a receptor site.
 149. The methodof claim 148, wherein the carrier is one selected from insulin,interleukin, thymopoietin or transferrin.
 150. The method of claim 148,wherein the at least one activatable pharmaceutical agent is coupled tothe carrier by a covalent bond.
 151. The method of claim 148, whereinthe at least one activatable pharmaceutical agent is coupled to thecarrier by non-covalent bond.
 152. The method of claim 148, wherein thereceptor site is one selected from nucleic acids of nucleated cells,antigenic sites on nucleated cells, or epitopes.
 153. The method ofclaim 133, wherein the at least one activatable pharmaceutical agent hasaffinity for a target cell.
 154. The method of claim 133, wherein the atleast one activatable pharmaceutical agent is capable of beingpreferentially absorbed by a target cell.
 155. The method of claim 133,wherein the predetermined cellular change is apoptosis in a target cell.156. The method of claim 133, wherein the at least one activatedpharmaceutical agent causes an auto-vaccine effect in the subject thatreacts with a targets cell.
 157. The method of claim 156, wherein theauto-vaccine effect is generated in a joint or lymph node.
 158. Themethod of claim 133, wherein the at least one activatable pharmaceuticalagent is a DNA intercalator or a halogenated derivative thereof. 159.The method of claim 133, wherein the initiation energy is one ofelectromagnetic energy, acoustic energy, or thermal energy.
 160. Themethod of claim 133, further comprising a blocking agent, wherein theblocking agent is capable of blocking uptake of the at least oneactivatable pharmaceutical agent prior to its activation.
 161. Themethod of claim 160, wherein the blocking agent is capable of slowingdown mitosis in non-target cells while allowing target cells to maintainan abnormal rate of mitosis.
 162. The method of claim 133, wherein saidat least one energy modulation agent is a single energy modulationagent, and is coupled to said at least one activatable pharmaceuticalagent.
 163. The method of claim 133, wherein a plurality of the energymodulation agents is administered, and wherein the enhanced or modifiedinitiation energy or the enhanced or modified initiation energy isconverted, through a cascade energy transfer between the plurality ofthe energy modulation agents, to an energy that activates the at leastone activatable pharmaceutical agent.
 164. The method of claim 133,wherein the at least one activatable pharmaceutical agent comprises anactive agent contained within a photocage, wherein upon exposure to areemitted energy by the at least one modulation agent as the activationenergy of the at least one activatable pharmaceutical agent, thephotocage disassociates from the active agent, rendering the activeagent available.
 165. The method of claim 133, wherein theplasmonics-active agent enhances the applied initiation energy from 10⁶to 10¹⁵ fold.
 166. The method of claim 133, wherein theplasmonics-active agent is at least one metal nanoparticle which isbound to the at least one energy modulation agent via a linker.
 167. Themethod of claim 133, wherein the energy modulation agent is at least oneselected from the group consisting of metals, quantum dots,semiconductor materials, scintillation and phosphor materials, materialsthat exhibit X-ray excited luminescence (XEOL), organic solids, metalcomplexes, inorganic solids, crystals, rare earth materials(lanthanides), polymers, scintillators, phosphor materials, andmaterials that exhibit excitonic properties.
 168. The method of claim138, wherein the plasmonics-active agent is a PEPST probe comprising atleast one plasmonics-active metal nanostructure, at least oneactivatable pharmaceutical agent, and at least one energy modulationagent.
 169. The method of claim 168, wherein the metal nanostructure isat least one selected from the group consisting of a nanosphere,nanorod, nanocube, nanopyramid, nanoshell, nanoshell cylinder, andmulti-layer nanoshells.
 170. The method of claim 133, wherein theplasmonics-active agent is a PEPST probe comprising multiple structuresfor different plasmonics activation regimes.
 171. The method of claim170, wherein said regimen is at least one selected from the groupconsisting of NIR and X ray.
 172. The method of claim 133, wherein theplasmonics-active agent is a PEPST probe comprising at least one metalnanosystem coupled with the energy modulation agent coupled and/or theactivatable pharmaceutical agent.
 173. The method of claim 172, whereinthe nanosystem is bound to the activatable pharmaceutical agent or theenergy modulation agent by a bond selected from the group consisting ofa chemical bond, a biochemical bond, a DNA bond, and an antigen-antibodybond.
 174. The method of claim 173, wherein the bond is a photo-liablebond.
 175. The method of claim 173, wherein the activatablepharmaceutical agent is released from the nanosystem inside a cell byphoton radiation.
 176. The method of claim 133, wherein theplasmonics-active agent is a PEPST probe comprising at least one metalnanostructure selected from the group consisting of a metalnanoparticle, a dielectric nanoparticle core covered with a metalnanocap, a spherical metal nanoshell covering a dielectric spheroidcore, a oblate metal nanoshell covering a dielectric spheroid core, ametal nanoparticle core covered with a dielectric nanoshell, a metalnanoshell with a protective coating layer, multi layer metal nanoshellscovering a dielectric spheroid core, multi-nanoparticle structures, ametal nanocube and a nanotriangle/nanoprism, and a metal cylinder. 177.The method of claim 133, wherein the plasmonics-active agent is a PEPSTprobe comprising a combination of metal nanoparticles covered by a layerof a dielectric material comprising the activatable pharmaceuticalagent, with the energy modulation agent which is bound to theactivatable pharmaceutical agent.
 178. The method of claim 133, whereinthe plasmonics-active agent is a PEPST probe, which enhances a XEOLlight emitted by the energy modulation agent irradiated by an X ray,wherein the enhanced XEOL light activates the activatable pharmaceuticalagent.
 179. The method of claim 133, wherein the plasmonics-active agentis a PEPST probe and which is irradiated by X ray to excite theactivatable pharmaceutical agent or the energy modulation agent coupledwith the activatable pharmaceutical agent.
 180. The method of claim 1,wherein the plasmonics-active agent is a PEPST probe comprisingplasmonics-active metal nanostructures, said PEPST probe is irradiatedby X ray to excite a surface plasmon in nanoparticles or subnanoparticleof a metal.
 181. The method of claim 180, wherein the nanoparticles orsubnanoparticle of the metal are gold or silver nanoparticles orsubnanoparticle.
 182. The method of claim 180, wherein the metalnanostructure is at least one selected from the group consisting of ananosphere, nanorod, nanocube, nanopyramid, nanoshell, and multi-layernanoshells.
 183. The method of claim 133, wherein the plasmonics-activeagent is a PEPST probe comprising the energy modulation agent coupledwith the activatable pharmaceutical agent and at least one bioreceptor.184. The method of claim 183, wherein the bioreceptor is at least oneselected from the group consisting an antibody/antigen, an enzyme, anucleic acid/DNA, a cellular structure/cell, and a biomimetic.
 185. Themethod of claim 133, comprising delivering to a target the activatablepharmaceutical agent using a target delivery system comprising theplasmonics-active agent comprising the activatable pharmaceutical agentand the energy modulation agent, wherein the plasmonics-active agent isa PEPST probe.
 186. The method of claim 133, wherein theplasmonics-active agent is a PEPST probe possessing plasmonicsphotospectral properties, biocompatibility, an improved drug payloaddelivery and passive targeting of metal nanoparticles.
 187. The methodof claim 133, wherein the plasmonics-active agent is a PEPST comprisingmetal nanoparticles and at least one biomolecule is immobilized on themetal nanoparticles.
 188. The method of claim 187, wherein thebiomolecule is the activatable pharmaceutical agent, the modulationagent, a drug, a protein, an enzyme, an antibody, DNA, or RNA.
 189. Themethod of claim 133, wherein the plasmonics-active agent is a PEPSTprobe comprising metal nanoparticles.
 190. The method of claim 189,wherein the metal nanoparticles are gold or silver nanoparticles. 191.The method of claim 189, wherein the metal nanoparticles enhance ormodify an X ray energy applied to the subject to excite the energymodulation agent which converts the enhanced or modulated X ray energyto an energy that activates the at least one activatable pharmaceuticalagent directly or via a cascade energy transfer between the plurality ofthe energy modulation agents.
 192. The method of claim 189, wherein themetal nanoparticles enhance or modify an energy emitted by themodulation agent which converts the initiation energy to the emittedenergy, said enhanced or modified emitted energy activates the at leastone activatable pharmaceutical agent.
 193. The method of claim 133,wherein the plasmonics-active agent is a PEPST probe comprising a chainof metal particles having the same of different size and coupled to oneanother, wherein the chain of the metal particles exhibit dual or multiplasmonics resonance modes.
 194. The method of claim 193, wherein thechain of particles are used for providing a plasmonics enhancement ofthe initiation energy and/or an energy emitted by the modulation agent.195. The method of claim 133, wherein the activatable pharmaceuticalagent is delivered to a target and is released from theplasmonics-active agent by photon radiation or ultrasound, and whereinthe plasmonics-active agent is a PEPST probe.
 196. The method of claim195, wherein the activatable pharmaceutical agent is released from theplasmonics-active agent comprising an antibody or antigen system coupledwith the activatable pharmaceutical agent.
 197. The method of claim 133,comprising delivering the activatable pharmaceutical agent to a targetusing liposomes.
 198. The method of claim 133, wherein theplasmonics-active agent is a PEPST probe comprising the activatablepharmaceutical agent which is encapsulated.
 199. The method of claim198, the activatable pharmaceutical agent or the activatablepharmaceutical agent and the modulation agent are encapsulated in acapsule comprising an ferritin and/or apoferritin compound.
 200. Themethod of claim 133, which uses ultrasound for a release of theactivatable pharmaceutical agent and photonic excitation of theactivatable pharmaceutical agent or an energy modulation agent coupledwith the activatable pharmaceutical agent, wherein the plasmonics-activeagent is a PEPST probe.
 201. The method of claim 133, the method furthercomprising using a drug delivery, tumor targeting, and/or drug releasingsystem for delivering and releasing the plasmonics-active agent and theactivatable pharmaceutical agent, wherein the plasmonics-active agent isa PEPST probe.
 202. The method of claim 133, wherein theplasmonics-active agent is a PEPST probe, wherein components of thePEPST probe are bound using conjugates, metals binding to organic andinorganic compounds, and biomolecules.
 203. The method of claim 133,wherein the plasmonics-active agent comprises a plasmonic-active metalnanoparticle bound to an excitation energy converter (EEC) materialwhich is bound to the activatable pharmaceutical agent, wherein the EECmaterial for exciton induced phototherapy (EIP) probes is optimizedbased on the exciton properties, and wherein the EEC material is theenergy modulation agent.
 204. The method of claim 133, wherein theplasmonics-active agent comprises an excitation energy converter (EEC)material, said method tunes an emission of the EEC material in excitoninduced phototherapy (EIP) probes to a wavelength capable of exiting theactivatable pharmaceutical agent using specific materials with specificexciton properties, wherein said EEC material can produce excitons undera radiative excitation, and wherein the EEC material is the energymodulation agent.
 205. The method of claim 204, wherein the EEC materialis the energy modulation agent, said EEC material is bound via a linkerto the activatable pharmaceutical agent.
 206. The method of claim 204,wherein the EEC material is the energy modulation agent, and theactivatable pharmaceutical agent is embedded in a shell around said EECmaterial.
 207. The method of claim 204, wherein the EEC material hasstructural defects that serve as traps for the excitation.
 208. Themethod of claim 204, wherein the EEC material comprises impurities ordopant molecules that serve as traps for the excitation.
 209. The methodof claim 204, wherein the initiation energy is X ray which istransformed to UV or visible photons by the EEC material.
 210. Themethod of claim 133, wherein the plasmonics-active agent is anexciton-plasmon enhanced phototherapy (EPEP) probe which comprises atleast one plasmonics-active metal nanostructure, at least oneexciton-generating energy modulation agent material, and the at leastone activatable pharmaceutical agent, wherein said metal nanostructureand said material produce exciton-plasmon (EPC) coupling.
 211. Themethod of claim 210, wherein the plasmonics-active metal nanostructureand the exciton-generating energy modulation agent material are coupledvia a spacer.
 212. The method of claim 210 or 211, whereinexciton-generating energy modulation agent material is coupled with theactivatable pharmaceutical agent via a linker.
 213. The method of claim210 or 211, wherein the activatable pharmaceutical agent and theplasmonics-active metal nanostructure are coupled via a linker.
 214. Themethod of claim 213, wherein the EPEP probe comprises a bioreceptor.215. The method of claim 214, wherein the bioreceptor is bound to theplasmonics-active metal nanostructure or to the plasmonics-active metalnanostructure covered with a nanoshell or a nanoshell cylinder made of adielectric material.
 216. The method of claim 210 or 211, wherein theplasmonics-active metal nanostructure is covered with a nanoshell madeof a dielectric material.
 217. The method of claim 210 or 211, whereinthe plasmonics-active metal nanostructure is a metal nanosphere, ananowire or a nanorod covered with a nanoshell or a nanoshell cylinderof a dielectric material.
 218. The method of claim 210, wherein the EPEPprobe comprises multiple metal nanowires coupled with theexciton-generating energy modulation agent material with or withoutspacers which can be the same or different, said exciton-generatingenergy modulation agent material is bound to the activatablepharmaceutical agent via a linker.
 219. The method of claim 210, whereinthe exciton-generating energy modulation agent material is amicroresonator.
 220. The method of claim 210, wherein theexciton-generating energy modulation agent material is at least oneselected from the group consisting of metals, quantum dots,semiconductor materials, scintillation and phosphor materials, materialsthat exhibit X-ray excited luminescence (XEOL), organic solids, metalcomplexes, inorganic solids, crystals, rare earth materials(lanthanides), polymers, scintillators, phosphor materials, andmaterials that exhibit excitonic properties.
 221. The method of claim210, wherein the metal nanostructure is at least one selected from thegroup consisting of a nano sphere, nanorod, nanocube, nanopyramid,nanoshell, nanoshell cylinder, and multi-layer nanoshells.
 222. Themethod of claim 210, wherein the EPEP probe comprises multiplestructures for different plasmonics activation regimes.
 223. The methodof claim 222, wherein said regime is at least one selected from thegroup consisting of NIR and X ray.
 224. The method of claim 210, whereinthe EPEP probe comprises at least one metal nanosystem coupled with theactivatable pharmaceutical agent, or with an energy modulation agentcoupled with the activatable pharmaceutical agent.
 225. The method ofclaim 224, wherein the nanosystem is bound to the activatablepharmaceutical agent or the energy modulation agent by a bond selectedfrom the group consisting of a chemical bond, a biochemical bond, a DNAbond, and an antigen-antibody bond.
 226. The method of claim 225,wherein the bond is a photo-liable bond.
 227. The method of claim 225,wherein the activatable pharmaceutical agent is released from thenanosystem inside a cell by photon radiation.
 228. The method of claim210, wherein the EPEP probe comprises at least one metal nanostructureselected from the group consisting of a metal nanoparticle, a dielectricnanoparticle core covered with a metal nanocap, a spherical metalnanoshell covering a dielectric spheroid core, a oblate metal nanoshellcovering a dielectric spheroid core, a metal nanoparticle core coveredwith a dielectric nanoshell, a metal nanoshell with a protective coatinglayer, multi layer metal nanoshells covering a dielectric spheroid core,multi-nanoparticle structures, a metal nanocube and ananotriangle/nanoprism, and a metal cylinder.
 229. The method of claim210, wherein the EPEP probe comprises a combination of metalnanoparticles with an energy modulation agent which is bound to theactivatable pharmaceutical agent, wherein the metal nanoparticles arecovered by a layer of a dielectric material comprising the activatablepharmaceutical agent.
 230. The method of claim 210, wherein the EPEPprobe enhances a XEOL light emitted by the exciton-generating energymodulation agent material irradiated by an X ray, wherein the enhancedXEOL light activates the activatable pharmaceutical agent.
 231. Themethod of claim 210, wherein the EPEP probe is irradiated by an X ray toexcite the activatable pharmaceutical agent or an energy modulationagent coupled with the activatable pharmaceutical agent.
 232. The methodof claim 210, wherein the EPEP probe comprising the plasmonics-activemetal nanostructures is irradiated by an X ray to excite a surfaceplasmon in nanoparticles or subnanoparticle of a metal.
 233. The methodof claim 232, wherein the nanoparticles or subnanoparticle of the metalare gold or silver nanoparticles or subnanoparticle.
 234. The method ofclaim 232, wherein the metal nanostructure is at least one selected fromthe group consisting of a nanosphere, nanorod, nanocube, nanopyramid,nanoshell, and multi-layer nanoshells.
 235. The method of claim 214,wherein the bioreceptor is at least one selected from the groupconsisting an antibody/antigen, an enzyme, a nucleic acid/DNA, acellular structure/cell, and a biomimetic.
 236. The method of claim 210,comprising delivering to a target the activatable pharmaceutical agentusing a target delivery system comprising the plasmonics-active metalnanostructure comprising the activatable pharmaceutical agent.
 237. Themethod of claim 210, wherein the EPEP probe possesses plasmonicsphotospectral properties, biocompatibility, an improved drug payloaddelivery and passive targeting of metal nanoparticles.
 238. The methodof claim 210, wherein at least one biomolecule is immobilized on themetal nanostructure.
 239. The method of claim 238, wherein thebiomolecule is at least one selected from the group consisting of theactivatable pharmaceutical agent, the modulation agent, a drug, aprotein, an enzyme, an antibody, and a nucleic acid.
 240. The method ofclaim 210, wherein the metal nanostructure is a gold or silvernanoparticle.
 241. The method of claim 210, wherein the metalnanostructure enhances or modifies an X ray energy applied to thesubject to excite the exciton-generating energy modulation agent whichconverts the enhanced or modulated X ray energy to an energy thatactivates the at least one activatable pharmaceutical agent directly orvia a cascade energy transfer between the plurality of the energymodulation agents.
 242. The method of claim 210, wherein the metalnanoparticle enhances or modifies an energy emitted by theexciton-generating energy modulation agent which converts the initiationenergy to the emitted energy, said enhanced or modified emitted energyactivates the at least one activatable pharmaceutical agent.
 243. Themethod of claim 210, wherein the metal nanostructure enhances ormodifies (i) an X ray energy applied to the subject to excite theexciton-generating energy modulation agent material which converts theenhanced or modulated X ray energy to an energy that activates the atleast one activatable pharmaceutical agent directly or via a cascadeenergy transfer between the plurality of the energy modulation agents,and (ii) an energy emitted by the exciton-generating energy modulationagent material which converts the enhanced or modified energy (i) to anenergy that activates the at least one activatable pharmaceutical agent.244. The method of claim 210, wherein (a) the exciton-generating energymodulation agent, (b) the activatable pharmaceutical agent, or (c) theexciton-generating energy modulation agent and the activatablepharmaceutical agent, are covered with a layer comprising theplasmonics-active metal nanostructure.
 245. The method of claim 210,wherein the EPEP probe comprises a chain of metal particles having thesame of different size and coupled to one another, wherein the chain ofthe metal particles exhibit dual or multi plasmonics resonance modes.246. The method of claim 245, wherein the chain of the particles is usedfor providing a plasmonics enhancement of the initiation energy and/oran energy emitted by the exciton-generating energy modulation agentmaterial.
 247. The method of claim 210, wherein the activatablepharmaceutical agent is delivered to a target and is released from theplasmonics-active metal nanostructure by photon radiation or ultrasound.248. The method of claim 247, wherein the activatable pharmaceuticalagent is released from the plasmonics-active metal nanostructurecomprising an antibody or antigen system coupled with the activatablepharmaceutical agent.
 249. The method of claim 210, comprisingdelivering the activatable pharmaceutical agent to a target usingliposomes.
 250. The method of claim 210, wherein the EPEP probecomprises the activatable pharmaceutical agent which is encapsulated.251. The method of claim 250, wherein the activatable pharmaceuticalagent or the activatable pharmaceutical agent and the exciton-generatingenergy modulation agent material are encapsulated in a capsulecomprising an ferritin and/or apoferritin compound.
 252. The method ofclaim 210, which uses ultrasound for a release of the activatablepharmaceutical agent and photonic excitation of the activatablepharmaceutical agent or the exciton-generating energy modulation agentmaterial coupled with the activatable pharmaceutical agent.
 253. Themethod of claim 133, the method further comprising using a drugdelivery, tumor targeting, and/or drug releasing system for deliveringand releasing the plasmonics-active agent and/or the activatablepharmaceutical agent, wherein the plasmonics-active agent is a EPEPprobe.
 254. The method of claim 133 or 210, wherein theplasmonics-active agent is a EPEP probe, wherein components of the EPEPprobe are bound using conjugates, metals binding to organic andinorganic compounds, and biomolecules.
 255. The method of claim 133 or210, wherein the plasmonics agent is a EPEP probe possessing EPIPproperties, biocompatibility, an improved drug payload delivery andpassive targeting of metal nanoparticles.
 256. A method for treating acell proliferation disorder in a subject, comprising: (1) administeringto the subject at least one activatable pharmaceutical agent that iscapable effecting a predetermined cellular change when activated and atleast one plasmonics-active agent, and (2) applying an initiation energyfrom an initiation energy source to the subject, wherein the initiationenergy is applied and activatable pharmaceutical agent upon theactivation produces insufficient singlet oxygen in the subject toproduce cell lysis, and wherein the plasmonics-active agent enhances ormodifies the applied initiation energy, such that the enhanced ormodified initiation energy activates the activatable pharmaceuticalagent in situ, thus causing the predetermined cellular change to occur,wherein said predetermined cellular change treats the cell proliferationrelated disorder.
 257. The method according to claim 256, wherein saidpredetermined cellular change treats the cell proliferation disorder bycausing an increase or decrease in cell proliferation rate of a targetcell.
 258. The method according to claim 256, wherein the amount ofsinglet oxygen production is less than 109 singlet oxygenmolecules/cell.
 259. The method according to claim 256, wherein theamount of singlet oxygen production is less than 0.32×10⁻³ mol/liter.260. The method according to claim 256, wherein the at least oneactivated pharmaceutical agent causes an auto-vaccine effect in thesubject that reacts with a target cell.
 261. The method of claim 260,wherein the auto-vaccine effect is generated in a joint or lymph node.262. The method according to claim 256, further comprising, prior tosaid applying of the initiation energy, administering to the subject atleast one energy modulation agent that converts the initiation energy toan energy that activates the at least one activatable pharmaceuticalagent.
 263. The method of claim 262, wherein a plurality of the energymodulation agents is administered, and wherein the initiation energy isconverted, through a cascade energy transfer between the plurality ofthe energy modulation agents, to the energy that activates the at leastone activatable pharmaceutical agent.
 264. The method of claim 262,wherein said at least one energy modulation agent is a single energymodulation agent, and is coupled to said at least one activatablepharmaceutical agent.
 265. The method of claim 256, wherein the at leastone activatable pharmaceutical agent comprises an active agent containedwithin a photocage, wherein upon exposure to said initiation energy, thephotocage disassociates from the active agent, rendering the activeagent available.
 266. The method of claim 262, wherein the at least oneactivatable pharmaceutical agent comprises an active agent containedwithin a photocage, wherein upon exposure to a reemitted energy by theat least one modulation agent as the activation energy of the at leastone activatable pharmaceutical agent, the photocage disassociates fromthe active agent, rendering the active agent available.
 267. The methodof claim 256, wherein the initiation energy source is selected from thegroup consisting of UV radiation, visible light, infrared radiation,x-rays, gamma rays, electron beams, phosphorescent compounds,chemiluminescent compounds, bioluminescent compounds, and light emittingenzymes.
 268. The method of claim 256, wherein the predeterminedcellular change is apoptosis in a target cell.
 269. The method of claim256, wherein the cell proliferation disorder is at least one memberselected from the consisting of cancer, bacterial infection, viralinfection, immune rejection response, autoimmune disorders, and aplasticconditions.
 270. The method of claim 256, wherein the at least oneactivatable pharmaceutical agent is a photoactivatable agent.
 271. Themethod of claim 256, wherein the at least one activatable pharmaceuticalagent is selected from psoralens, pyrene cholesteryloleate, acridine,porphyrin, fluorescein, rhodamine, 16-diazorcortisone, ethidium,transition metal complexes of bleomycin, transition metal complexes ofdeglycobleomycin organoplatinum complexes, alloxazines, vitamin Ks,vitamin L, vitamin metabolites, vitamin precursors, naphthoquinones,naphthalenes, naphthols and derivatives thereof having planar molecularconformations, porphorinporphyrins, dyes and phenothiazine derivatives,coumarins, quinolones, quinones, and anthroquinones.
 272. The method ofclaim 271, wherein the at least one activatable pharmaceutical agent isa psoralen, a coumarin, a porphyrin, or a derivative thereof.
 273. Themethod of claim 271, wherein the at least one activatable pharmaceuticalagent is 8-MOP or AMT.
 274. The method of claim 256, wherein the atleast one activatable pharmaceutical agent is one selected from7,8-dimethyl-10-ribityl, isoalloxazine, 7,8,10-trimethylisoalloxazine,7,8-dimethylalloxazine, isoalloxazine-adenine dinucleotide, alloxazinemononucleotide, aluminum (III) phthalocyanine tetrasulonate,hematophorphyrin, and phthadocyanine.
 275. The method of claim 256,wherein the at least one activatable pharmaceutical agent is coupled toa carrier that is capable of binding to a receptor site.
 276. The methodof claim 275, wherein the carrier is one selected from insulin,interleukin, thymopoietin or transferrin.
 277. The method of claim 275,wherein the at least one activatable pharmaceutical agent is coupled tothe carrier by a covalent bond.
 278. The method of claim 275, whereinthe at least one activatable pharmaceutical agent is coupled to thecarrier by a non-covalent bond.
 279. The method of claim 275, whereinthe receptor site is one selected from nucleic acids of nucleated cells,antigenic sites on nucleated cells, or epitopes.
 280. The method ofclaim 256, wherein the at least one activatable pharmaceutical agent hasaffinity for a target cell.
 281. The method of claim 256, wherein the atleast one activatable pharmaceutical agent is capable of beingpreferentially absorbed by a target cell.
 282. The method of claim 256,wherein the at least one activatable pharmaceutical agent is a DNAintercalator or a halogenated derivative thereof.
 283. The method ofclaim 256, wherein the plasmonics-active agent enhances the appliedinitiation energy from 10⁶ to 10¹⁵ fold.
 284. The method of claim 262,wherein the plasmonics-active agent is at least one metal nanoparticlewhich is bound to the at least one energy modulation agent via a linker.285. The method of claim 262, wherein the energy modulation agent is atleast one selected from the group consisting of metals, quantum dots,semiconductor materials, scintillation and phosphor materials, materialsthat exhibit X-ray excited luminescence (XEOL), organic solids, metalcomplexes, inorganic solids, crystals, rare earth materials(lanthanides), polymers, scintillators, phosphor materials, andmaterials that exhibit excitonic properties.
 286. The method of claim256, wherein the plasmonics-active agent is a PEPST probe comprising atleast one plasmonics-active metal nanostructure, at least oneactivatable pharmaceutical agent, and at least one energy modulationagent.
 287. The method of claim 286, wherein the metal nanostructure isat least one selected from the group consisting of a nanosphere,nanorod, nanocube, nanopyramid, nanoshell, nanoshell cylinder, andmulti-layer nanoshells.
 288. The method of claim 256, wherein theplasmonics-active agent is a PEPST probe comprising multiple structuresfor different plasmonics activation regimes.
 289. The method of claim288, wherein said regime is at least one selected from the groupconsisting of NIR and X ray.
 290. The method of claim 262, wherein theplasmonics-active agent is a PEPST probe comprising at least one metalnanosystem coupled with the energy modulation agent coupled and/or theactivatable pharmaceutical agent.
 291. The method of claim 290, whereinthe nanosystem is bound to the activatable pharmaceutical agent or theenergy modulation agent by a bond selected from the group consisting ofa chemical bond, a biochemical bond, a DNA bond, and an antigen-antibodybond.
 292. The method of claim 291, wherein the bond is a photo-liablebond.
 293. The method of claim 291, wherein the activatablepharmaceutical agent is released from the nanosystem inside a cell byphoton radiation.
 294. The method of claim 256, wherein theplasmonics-active agent is a PEPST probe comprising at least one metalnanostructure selected from the group consisting of a metalnanoparticle, a dielectric nanoparticle core covered with a metalnanocap, a spherical metal nanoshell covering a dielectric spheroidcore, a oblate metal nanoshell covering a dielectric spheroid core, ametal nanoparticle core covered with a dielectric nanoshell, a metalnanoshell with a protective coating layer, multi layer metal nanoshellscovering a dielectric spheroid core, multi-nanoparticle structures, ametal nanocube and a nanotriangle/nanoprism, and a metal cylinder. 295.The method of claim 262, wherein the plasmonics-active agent is a PEPSTprobe comprising a combination of metal nanoparticles covered by a layerof a dielectric material comprising the activatable pharmaceuticalagent, with the energy modulation agent which is bound to theactivatable pharmaceutical agent.
 296. The method of claim 262, whereinthe plasmonics-active agent is a PEPST probe, which enhances a XEOLlight emitted by the energy modulation agent irradiated by an X ray,wherein the enhanced XEOL light activates the activatable pharmaceuticalagent.
 297. The method of claim 262, wherein the plasmonics-active agentis a PEPST probe and which is irradiated by X ray to excite theactivatable pharmaceutical agent or the energy modulation agent coupledwith the activatable pharmaceutical agent.
 298. The method of claim 256,wherein the plasmonics-active agent is a PEPST probe comprisingplasmonics-active metal nanostructures, said PEPST probe is irradiatedby X ray to excite a surface plasmon in nanoparticles or subnanoparticleof a metal.
 299. The method of claim 298, wherein the nanoparticles orsubnanoparticle of the metal are gold or silver nanoparticles orsubnanoparticle.
 300. The method of claim 256, wherein theplasmonics-active agent is a PEPST probe comprising a metalnanostructure, wherein the metal nanostructure is at least one selectedfrom the group consisting of a nanosphere, nanorod, nanocube,nanopyramid, nanoshell, and multi-layer nanoshells.
 301. The method ofclaim 262, wherein the plasmonics-active agent is a PEPST probecomprising the energy modulation agent coupled with the activatablepharmaceutical agent and at least one bioreceptor.
 302. The method ofclaim 301, wherein the bioreceptor is at least one selected from thegroup consisting an antibody/antigen, an enzyme, a nucleic acid/DNA, acellular structure/cell, and a biomimetic.
 303. The method of claim 256,comprising delivering to a target the activatable pharmaceutical agentusing a target delivery system comprising the plasmonics-active agentcomprising the activatable pharmaceutical agent and, optionally, anenergy modulation agent, wherein the plasmonics-active agent is a PEPSTprobe.
 304. The method of claim 256, wherein the plasmonics-active agentis a PEPST probe possessing plasmonics photospectral properties,biocompatibility, an improved drug payload delivery and passivetargeting of metal nanoparticles.
 305. The method of claim 256, whereinthe plasmonics-active agent is a PEPST probe comprising metalnanoparticles and at least one biomolecule is immobilized on the metalnanoparticles.
 306. The method of claim 305, wherein the biomolecule isthe activatable pharmaceutical agent, the modulation agent, a drug, aprotein, an enzyme, an antibody, DNA, or RNA.
 307. The method of claim256, wherein the plasmonics-active agent is a PEPST probe comprisingmetal nanoparticles, said metal nanoparticles are gold or silvernanoparticles.
 308. The method of claim 262, wherein metal nanoparticlesenhance or modify an X ray energy applied to the subject to excite theenergy modulation agent which converts the enhanced or modified X rayenergy to an energy that activates the at least one activatablepharmaceutical agent directly or via a cascade energy transfer betweenthe plurality of the energy modulation agents.
 309. The method of claim262, wherein metal nanoparticles enhance or modify an energy emitted bythe modulation agent which converts the initiation energy to the emittedenergy, said enhanced or modified emitted energy activates the at leastone activatable pharmaceutical agent.
 310. The method of claim 256,wherein the plasmonics-active agent is a PEPST probe comprising a chainof metal particles having the same of different size and coupled to oneanother, wherein the chain of the metal particles exhibit dual or multiplasmonics resonance modes.
 311. The method of claim 310, wherein thechain of particles are used for providing a plasmonics enhancement ofthe initiation energy and/or an energy emitted by the modulation agent.312. The method of claim 256, wherein the activatable pharmaceuticalagent is delivered to a target and is released from theplasmonics-active agent by photon radiation or ultrasound, and whereinthe plasmonics-active agent is a PEPST probe.
 313. The method of claim262, wherein the activatable pharmaceutical agent is delivered to atarget and is released from the energy modulation agent couples with theplasmonics-active agent by photon radiation or ultrasound, and whereinthe plasmonics-active agent is a PEPST probe.
 314. The method of claim312, wherein the activatable pharmaceutical agent is released from theplasmonics-active agent comprising an antibody or antigen system coupledwith the activatable pharmaceutical agent.
 315. The method of claim 256,comprising delivering the activatable pharmaceutical agent to a targetusing liposomes.
 316. The method of claim 256, wherein theplasmonics-active agent is a PEPST probe comprising the activatablepharmaceutical agent which is encapsulated.
 317. The method of claim316, the activatable pharmaceutical agent and, optionally, a modulationagent are encapsulated in a capsule comprising an ferritin and/orapoferritin compound.
 318. The method of claim 256, which usesultrasound for a release of the activatable pharmaceutical agent andphotonic excitation of the activatable pharmaceutical agent or an energymodulation agent coupled with the activatable pharmaceutical agent,wherein the plasmonics-active agent is a PEPST probe.
 319. The method ofclaim 256, wherein the plasmonics-active agent is a PEPST probe. 320.The method of claim 256, the method further comprising using a drugdelivery, tumor targeting, and/or drug releasing system for deliveringand releasing the plasmonics-active agent and/or the activatablepharmaceutical agent, wherein the plasmonics-active agent is a PEPSTprobe.
 321. The method of claim 256, wherein the plasmonics-active agentis a PEPST probe, wherein components of the PEPST probe are bound usingconjugates, metals binding to organic and inorganic compounds, andbiomolecules.
 322. The method of claim 256, wherein theplasmonics-active agent comprises a plasmonic-active metal nanoparticlebound to an excitation energy converter (EEC) material which is bound tothe activatable pharmaceutical agent, wherein the EEC material forexciton induced phototherapy (EIP) probes is optimized based on theexciton properties, and wherein the EEC material is the energymodulation agent.
 323. The method of claim 256, wherein theplasmonics-active agent comprises an excitation energy converter (EEC)material, said method tunes an emission of the EEC material in excitoninduced phototherapy (EIP) probes to a wavelength capable of exiting theactivatable pharmaceutical agent using specific materials with specificexciton properties, wherein said EEC material can produce excitons undera radiative excitation, wherein the EEC material is the energymodulation agent.
 324. The method of claim 322, wherein the EEC materialis the energy modulation agent, said EEC material is bound via a linkerto the activatable pharmaceutical agent.
 325. The method of claim 322,wherein the EEC material is the energy modulation agent, and theactivatable pharmaceutical agent is embedded in a shell around said EECmaterial.
 326. The method of claim 322, wherein the EEC material hasstructural defects that serve as traps for the excitation.
 327. Themethod of claim 322, wherein the EEC material comprises impurities ordopant molecules that serve as traps for the excitation.
 328. The methodof claim 322, wherein the initiation energy is X ray which istransformed to UV or visible photons by the EEC material.
 329. Themethod of claim 256, wherein the plasmonics-active agent is anexciton-plasmon enhanced phototherapy (EPEP) probe which comprises atleast one plasmonics-active metal nanostructure, at least oneexciton-generating energy modulation agent material, and the at leastone activatable pharmaceutical agent, wherein said metal nanostructureand said material produce exciton-plasmon (EPC) coupling.
 330. Themethod of claim 323, wherein the plasmonics-active metal nanostructureand the exciton-generating energy modulation agent material are coupledvia a spacer.
 331. The method of claim 323, wherein exciton-generatingenergy modulation agent material is coupled with the activatablepharmaceutical agent via a linker.
 332. The method of claim 323, whereinthe activatable pharmaceutical agent and the plasmonics-active metalnanostructure are coupled via a linker.
 333. The method of claim 323,wherein the EPEP probe comprises a bioreceptor.
 334. The method of claim333, wherein the bioreceptor is bound to the plasmonics-active metalnanostructure or to the plasmonics-active metal nanostructure coveredwith a nanoshell or a nanoshell cylinder made of a dielectric material.335. The method of claim 323, wherein the plasmonics-active metalnanostructure is covered with a nanoshell made of a dielectric material.336. The method of claim 323, wherein the plasmonics-active metalnanostructure is a metal nanosphere, a nanowire or a nanorod coveredwith a nanoshell or a nanoshell cylinder of a dielectric material. 337.The method of claim 323, wherein the EPEP probe comprises multiple metalnanowires coupled with the exciton-generating energy modulation agentmaterial with or without spacers which can be the same or different,said exciton-generating energy modulation agent material is bound to theactivatable pharmaceutical agent via a linker.
 338. The method of claim323, wherein the exciton-generating energy modulation agent material isa microresonator.
 339. The method of claim 323, wherein theexciton-generating energy modulation agent material is at least oneselected from the group consisting of metals, quantum dots,semiconductor materials, scintillation and phosphor materials, materialsthat exhibit X-ray excited luminescence (XEOL), organic solids, metalcomplexes, inorganic solids, crystals, rare earth materials(lanthanides), polymers, scintillators, phosphor materials, andmaterials that exhibit excitonic properties.
 340. The method of claim323, wherein the metal nanostructure is at least one selected from thegroup consisting of a nanosphere, nanorod, nanocube, nanopyramid,nanoshell, nanoshell cylinder, and multi-layer nanoshells.
 341. Themethod of claim 323, wherein the EPEP probe comprises multiplestructures for different plasmonics activation regimes.
 342. The methodof claim 341, wherein said regime is at least one selected from thegroup consisting of NIR and X ray.
 343. The method of claim 323, whereinthe EPEP probe comprises at least one metal nanosystem coupled with theactivatable pharmaceutical agent, or with an energy modulation agentcoupled with the activatable pharmaceutical agent.
 344. The method ofclaim 343, wherein the nanosystem is bound to the activatablepharmaceutical agent or the energy modulation agent by a bond selectedfrom the group consisting of a chemical bond, a biochemical bond, a DNAbond, and an antigen-antibody bond.
 345. The method of claim 344,wherein the bond is a photo-liable bond.
 346. The method of claim 344,wherein the activatable pharmaceutical agent is released from thenanosystem inside a cell by photon radiation.
 347. The method of claim323, wherein the EPEP probe comprises at least one metal nanostructureselected from the group consisting of a metal nanoparticle, a dielectricnanoparticle core covered with a metal nanocap, a spherical metalnanoshell covering a dielectric spheroid core, a oblate metal nanoshellcovering a dielectric spheroid core, a metal nanoparticle core coveredwith a dielectric nanoshell, a metal nanoshell with a protective coatinglayer, multi layer metal nanoshells covering a dielectric spheroid core,multi-nanoparticle structures, a metal nanocube and ananotriangle/nanoprism, and a metal cylinder.
 348. The method of claim323, wherein the EPEP probe comprises a combination of metalnanoparticles with an energy modulation agent which is bound to theactivatable pharmaceutical agent, wherein the metal nanoparticles arecovered by a layer of a dielectric material comprising the activatablepharmaceutical agent.
 349. The method of claim 323, wherein the EPEPprobe enhances a XEOL light emitted by the exciton-generating energymodulation agent material irradiated by an X ray, wherein the enhancedXEOL light activates the activatable pharmaceutical agent.
 350. Themethod of claim 323, wherein the EPEP probe is irradiated by an X ray toexcite the activatable pharmaceutical agent or an energy modulationagent coupled with the activatable pharmaceutical agent.
 351. The methodof claim 323, wherein the EPEP probe comprising the plasmonics-activemetal nanostructures is irradiated by an X ray to excite a surfaceplasmon in nanoparticles or subnanoparticle of a metal.
 352. The methodof claim 351, wherein the nanoparticles or subnanoparticle of the metalare gold or silver nanoparticles or subnanoparticle.
 353. The method ofclaim 351, wherein the metal nanostructure is at least one selected fromthe group consisting of a nanosphere, nanorod, nanocube, nanopyramid,nanoshell, and multi-layer nanoshells.
 354. The method of claim 333,wherein the bioreceptor is at least one selected from the groupconsisting an antibody/antigen, an enzyme, a nucleic acid/DNA, acellular structure/cell, and a biomimetic.
 355. The method of claim 323,comprising delivering to a target the activatable pharmaceutical agentusing a target delivery system comprising the plasmonics-active metalnanostructure comprising the activatable pharmaceutical agent.
 356. Themethod of claim 323, wherein the EPEP probe possesses plasmonicsphotospectral properties, biocompatibility, an improved drug payloaddelivery and passive targeting of metal nanoparticles.
 357. The methodof claim 323, wherein at least one biomolecule is immobilized on themetal nanostructure.
 358. The method of claim 357, wherein thebiomolecule is at least one selected from the group consisting of theactivatable pharmaceutical agent, the modulation agent, a drug, aprotein, an enzyme, an antibody, and a nucleic acid.
 359. The method ofclaim 323, wherein the metal nanostructure is a gold or silvernanoparticle.
 360. The method of claim 323, wherein the metalnanostructure enhances or modulates an X ray energy applied to thesubject to excite the exciton-generating energy modulation agent whichconverts the enhanced or modulated X ray energy to an energy thatactivates the at least one activatable pharmaceutical agent directly orvia a cascade energy transfer between the plurality of the energymodulation agents.
 361. The method of claim 323, wherein the metalnanoparticle enhances or modifies an energy emitted by theexciton-generating energy modulation agent which converts the initiationenergy to the emitted energy, said enhanced or modified emitted energyactivates the at least one activatable pharmaceutical agent.
 362. Themethod of claim 323, wherein the metal nanostructure enhances ormodifies (i) an X ray energy applied to the subject to excite theexciton-generating energy modulation agent material which converts theenhanced or modulated X ray energy to an energy that activates the atleast one activatable pharmaceutical agent directly or via a cascadeenergy transfer between the plurality of the energy modulation agents,and (ii) an energy emitted by the exciton-generating energy modulationagent material which converts the enhanced or modulated energy (i) to anenergy that activates the at least one activatable pharmaceutical agent.363. The method of claim 323, wherein (a) the exciton-generating energymodulation agent, (b) the activatable pharmaceutical agent, or (c) theexciton-generating energy modulation agent and the activatablepharmaceutical agent, are covered with a layer comprising theplasmonics-active metal nanostructure.
 364. The method of claim 323,wherein the EPEP probe comprises a chain of metal particles having thesame of different size and coupled to one another, wherein the chain ofthe metal particles exhibit dual or multi plasmonics resonance modes.365. The method of claim 364, wherein the chain of the particles is usedfor providing a plasmonics enhancement of the initiation energy and/oran energy emitted by the exciton-generating energy modulation agentmaterial.
 366. The method of claim 323, wherein the activatablepharmaceutical agent is delivered to a target and is released from theplasmonics-active metal nanostructure by photon radiation or ultrasound.367. The method of claim 366, wherein the activatable pharmaceuticalagent is released from the plasmonics-active metal nanostructurecomprising an antibody or antigen system coupled with the activatablepharmaceutical agent.
 368. The method of claim 323, comprisingdelivering the activatable pharmaceutical agent to a target usingliposomes.
 369. The method of claim 323, wherein the EPEP probecomprises the activatable pharmaceutical agent which is encapsulated.370. The method of claim 369, wherein the activatable pharmaceuticalagent or the activatable pharmaceutical agent and the exciton-generatingenergy modulation agent material are encapsulated in a capsulecomprising an ferritin and/or apoferritin compound.
 371. The method ofclaim 323, which uses ultrasound for a release of the activatablepharmaceutical agent and photonic excitation of the activatablepharmaceutical agent or the exciton-generating energy modulation agentmaterial coupled with the activatable pharmaceutical agent.
 372. Themethod of claim 323, the method further comprising using a drugdelivery, tumor targeting, and/or drug releasing system for deliveringand releasing the plasmonics-active agent and the activatablepharmaceutical agent, wherein the plasmonics-active agent is a EPEPprobe.
 373. The method of claim 323, wherein the plasmonics-active agentis a EPEP probe, wherein components of the EPEP probe are bound usingconjugates, metals binding to organic and inorganic compounds, andbiomolecules.
 374. The method of claim 323, wherein the plasmonics agentis a EPEP probe possessing EPIP properties, biocompatibility, animproved drug payload delivery and passive targeting of metalnanoparticles.
 375. A method for generating an autovaccine for asubject, comprising: (1) providing a population of target cells; (2)treating the target cells ex vivo in an environment separate andisolated from the subject with an activatable pharmaceutical agent andwith a plasmonics-active agent; (3) expose the treated target cells toan energy source; (4) exciting the plasmonics-active agent and theactivatable pharmaceutical agent with an initiation energy, wherein theplasmonics-active agent enhances or modifies the initiation energy sothat to activate the activatable pharmaceutical agent with the enhancedor modified energy to induce a predetermined cellular change in thetarget cells; and (5) returning the thus changed cells back to thesubject to induce in the subject an autovaccine effect against thetarget cell, wherein the changed cells act as an autovaccine and theenergy source is selected from UV radiation, visible light, infraredradiation, x-rays, gamma rays, an electron beam, microwaves and radiowaves.
 376. The method of claim 375, wherein the at least oneactivatable pharmaceutical agent is selected from psoralens, pyrenecholesteryloleate, acridine, porphyrin, fluorescein, rhodamine,16-diazorcortisone, ethidium, transition metal complexes of bleomycin,transition metal complexes of deglycobleomycin organoplatinum complexes,alloxazines, vitamin Ks, vitamin L, vitamin metabolites, vitaminprecursors, naphthoquinones, naphthalenes, naphthols and derivativesthereof having planar molecular conformations, porphorinporphyrins, dyesand phenothiazine derivatives, coumarins, quinolones, quinones, andanthroquinones.
 377. The method of claim 376, wherein the at least oneactivatable pharmaceutical agent is a psoralen, a coumarin, a porphyrin,or a derivative thereof.
 378. The method of claim 377, wherein the atleast one activatable pharmaceutical agent is 8-MOP or AMT.
 379. Themethod of claim 375, wherein the at least one activatable pharmaceuticalagent is one selected from 7,8-dimethyl-10-ribityl, isoalloxazine,7,8,10-trimethylisoalloxazine, 7,8-dimethylalloxazine,isoalloxazine-adenine dinucleotide, alloxazine mononucleotide, aluminum(III) phthalocyanine tetrasulonate, hematophorphyrin, andphthadocyanine.
 380. The method of claim 375, further comprising:fractionating the apoptic cells and testing the fractions forauto-vaccine effect of each isolated component to determine thecomponent(s) associated with auto-vaccine before returning components tothe subject.
 381. The method of claim 376, wherein the predeterminedcellular change is apoptosis in a target cell affected by the cellproliferation disorder.
 382. The method of claim 375, wherein theplasmonics-active agent is a PEPST probe.
 383. The method of claim 375,wherein the plasmonics-active agent is a EPEP probe.
 384. A system forproducing an auto-vaccine in a subject, comprising: at least oneactivatable pharmaceutical agent that is capable of inducing apredetermined cellular change in a target cell in said subject; at leastone plasmonics-active agent capable of enhancing or modifying aninitiation energy; means for placing said at least one activatablepharmaceutical agent and said at least one plasmonics-active agent insaid subject; and an initiation energy source to provide the initiationenergy enhanced or modified by the plasmonics-active agent capable ofactivating the at least one activatable pharmaceutical agent in saidtarget cell, wherein activation is either direct or indirect.
 385. Thesystem of claim 384, wherein the predetermined cellular change isapoptosis in a target cell.
 386. The system of claim 384, wherein theinitiation energy is capable of directly activating the at least oneactivatable pharmaceutical agent.
 387. The system of claim 384, whereinthe initiation energy is UV, visible light, IR or NIR, x-rays, gammarays, an electron beam, microwaves or radio waves.
 388. The system ofclaim 384, further comprising at least one energy modulation agent forconverting the initiation energy to an energy that activate the at leastone activatable pharmaceutical agent.
 389. The system of claim 388,wherein the at least one energy modulation agent upgrades the energy ofthe initiation source and re-emits as an activation energy for the atleast one activatable pharmaceutical agent.
 390. The system of claim388, wherein the initiation source energy is x-rays, gamma rays, anelectron beam, microwaves or radio waves.
 391. The system of claim 388,wherein the at least one energy modulation agent downgrades the energyof the initiation source and re-emits as an activation energy for the atleast one activatable pharmaceutical agent.
 392. The system of claim391, wherein the initiation source energy is UV radiation, visiblelight, infrared radiation, x-rays, gamma rays, an electron beam,microwaves or radio waves.
 393. The system of claim 388, wherein said atleast one energy modulation agent is a single energy modulation agent,and is coupled to said at least one activatable pharmaceutical agent.394. The system of claim 388, comprising a plurality of the energymodulation agents for emitting an energy to the at least one activatablepharmaceutical agent, wherein the initiation energy is converted,through a cascade energy transfer between the plurality of energymodulation agents, to the energy that activates the at least oneactivatable pharmaceutical agent.
 395. The system of claim 384, whereinthe at least one activatable pharmaceutical agent is a photoactivatableagent.
 396. The system of claim 384, wherein the at least oneactivatable pharmaceutical agent is selected from psoralens, pyrenecholesteryloleate, acridine, porphyrin, fluorescein, rhodamine,16-diazorcortisone, ethidium, transition metal complexes of bleomycin,transition metal complexes of deglycobleomycin organoplatinum complexes,alloxazines, vitamin Ks, vitamin L, vitamin metabolites, vitaminprecursors, naphthoquinones, naphthalenes, naphthols and derivativesthereof having planar molecular conformations, porphorinporphyrins, dyesand phenothiazine derivatives, coumarins, quinolones, quinones, andanthroquinones.
 397. The system of claim 396, wherein the at least oneactivatable pharmaceutical agent is a psoralen, a coumarin, a porphyrin,or a derivative thereof.
 398. The system of claim 397, wherein the atleast one activatable pharmaceutical agent is 8-MOP or AMT.
 399. Thesystem of claim 384, wherein the at least one activatable pharmaceuticalagent is one selected from 7,8-dimethyl-10-ribityl, isoalloxazine,7,8,10-trimethylisoalloxazine, 7,8-dimethylalloxazine,isoalloxazine-adenine dinucleotide, alloxazine mononucleotide, aluminum(III) phthalocyanine tetrasulonate, hematophorphyrin, andphthadocyanine.
 400. The system of claim 384, wherein theplasmonics-active agent is a PEPST probe.
 401. The system of claim 384,wherein the plasmonics-active agent is a EPEP probe.
 402. The system ofclaim 384, wherein the at least one activatable pharmaceutical agent iscoupled to a carrier that is capable of binding to a receptor site. 403.The system of claim 402, wherein the carrier is one selected frominsulin, interleukin, thymopoietin or transferrin.
 404. The system ofclaim 402, wherein the receptor site is one selected from nucleic acidsof nucleated cells, antigenic sites on nucleated cells, or epitopes.405. The system of claim 384, wherein the at least one activatablepharmaceutical agent has affinity for a target cell.
 406. The system ofclaim 384, wherein the at least one activatable pharmaceutical agent iscapable of being preferentially absorbed by a target cell.
 407. Thesystem of claim 384, wherein the at least one activated pharmaceuticalagent causes an auto-vaccine effect in the subject that reacts with atarget cell.
 408. The system of claim 407, wherein the auto-vaccineeffect is generated in a joint or lymph node.
 409. The system of claim384, wherein the at least one activatable pharmaceutical agent is a DNAintercalator or a halogenated derivative thereof.
 410. The system ofclaim 388, wherein the at least one activatable pharmaceutical agentcomprises an active agent contained within a photocage, wherein uponexposure to said enhanced or modulated initiation energy, the photocagedisassociates from the active agent, rendering the active agentavailable.
 411. The system of claim 388, wherein the at least oneactivatable pharmaceutical agent comprises an active agent containedwithin a photocage, wherein upon exposure to said reemitted energy bythe modulation agent or the enhanced or modified by the photonics-activeagent re-emitted energy as the activation energy the at least oneactivatable pharmaceutical agent, the photocage disassociates from theactive agent, rendering the active agent available.
 412. Apharmaceutical composition for treating a cell proliferation disorder,comprising: at least one activatable pharmaceutical agent capable ofactivation and of causing a predetermined cellular change; at least oneplasmonics-active agent capable of enhancing or modifying energy; and apharmaceutically acceptable carrier.
 413. The pharmaceutical compositionof claim 412, further comprising at least one additive having acomplementary therapeutic or diagnostic effect, wherein said additive isat least one member selected from the group consisting of antioxidants,adjuvants, chemical energy sources, and combinations thereof.
 414. Thepharmaceutical composition of claim 412, wherein the at least oneactivatable pharmaceutical agent is a photoactivatable agent.
 415. Thepharmaceutical composition of claim 412, wherein the at least oneactivatable pharmaceutical agent is selected from psoralens, pyrenecholesteryloleate, acridine, porphyrin, fluorescein, rhodamine,16-diazorcortisone, ethidium, transition metal complexes of bleomycin,transition metal complexes of deglycobleomycin organoplatinum complexes,alloxazines, vitamin Ks, vitamin L, vitamin metabolites, vitaminprecursors, naphthoquinones, naphthalenes, naphthols and derivativesthereof having planar molecular conformations, porphorinporphyrins, dyesand phenothiazine derivatives, coumarins, quinolones, quinones, andanthroquinones.
 416. The pharmaceutical composition of claim 415,wherein the at least one activatable pharmaceutical agent is a psoralen,a coumarin, a porphyrin, or a derivative thereof.
 417. Thepharmaceutical composition of claim 415, wherein the at least oneactivatable pharmaceutical agent is 8-MOP or AMT.
 418. Thepharmaceutical composition of claim 412, wherein the at least oneactivatable pharmaceutical agent is one selected from7,8-dimethyl-10-ribityl, isoalloxazine, 7,8,10-trimethylisoalloxazine,7,8-dimethylalloxazine, isoalloxazine-adenine dinucleotide, alloxazinemononucleotide, aluminum (III) phthalocyanine tetrasulonate,hematophorphyrin, and phthadocyanine.
 419. The pharmaceuticalcomposition of claim 412, wherein the at least one activatablepharmaceutical agent is coupled to a carrier that is capable of bindingto a receptor site.
 420. The pharmaceutical composition of claim 419,wherein the carrier is one selected from insulin, interleukin,thymopoietin or transferring.
 421. The pharmaceutical composition ofclaim 419, wherein the at least one activatable pharmaceutical agent iscoupled to the carrier by a covalent bond.
 422. The pharmaceuticalcomposition of claim 419, wherein the at least one activatablepharmaceutical agent is coupled to the carrier by a non-covalent bond.423. The pharmaceutical composition of claim 419, wherein the receptorsite is one selected from nucleic acids of nucleated cells, antigenicsites on nucleated cells, or epitopes.
 424. The pharmaceuticalcomposition of claim 419, wherein the at least one activatablepharmaceutical agent has affinity for a target cell.
 425. Thepharmaceutical composition of claim 412, wherein the at least oneactivatable pharmaceutical agent is capable of being preferentiallyabsorbed by a target cell.
 426. The pharmaceutical composition of claim414, wherein the at least one activated pharmaceutical agent causes anauto-vaccine effect in the subject that reacts with a target cell. 427.The pharmaceutical composition of claim 412, wherein the at least oneactivatable pharmaceutical agent is a DNA intercalator or a halogenatedderivative thereof.
 428. The pharmaceutical composition of claim 412,wherein the predetermined cellular change is apoptosis in a target cell.429. The pharmaceutical composition of claim 412, further comprising atleast one energy modulation agent capable of activating the at least oneactivatable pharmaceutical agent when energized.
 430. The pharmaceuticalcomposition of claim 429, wherein said at least one energy modulationagent is a single energy modulation agent, and is coupled to said atleast one activatable pharmaceutical agent.
 431. The pharmaceuticalcomposition of claim 429, comprising a plurality of the energymodulation agents capable of converting the initiation energy ofenhanced or modified initiation energy by the photonics-active agent,through a cascade energy transfer between the plurality of energymodulation agents, to an energy that activates the at least oneactivatable pharmaceutical agent.
 432. The pharmaceutical composition ofclaim 412, wherein the at least one activatable pharmaceutical agentcomprises an active agent contained within a photocage, wherein uponexposure to said initiation energy source, the photocage disassociatesfrom the active agent, rendering the active agent available.
 433. Thepharmaceutical composition of claim 429, wherein the at least oneactivatable pharmaceutical agent comprises an active agent containedwithin a photocage, wherein upon exposure to a reemitted energy by themodulation agent as an activation energy of the at least one activatablepharmaceutical agent, the photocage disassociates from the active agent,rendering the active agent available.
 434. The pharmaceuticalcomposition of claim 412, wherein the at least one additive is achemical energy source.
 435. The pharmaceutical composition of claim434, wherein the chemical energy source is a member selected from thegroup consisting of phosphorescent compounds, chemiluminescentcompounds, bioluminescent compounds and light emitting enzymes.
 436. Thepharmaceutical composition of claim 412, wherein the photonics-activeagent is a PEPST probe.
 437. The pharmaceutical composition of claim412, wherein the photonics-active agent is a EPEP probe.
 438. A methodfor causing apoptosis in a subject in vivo, comprising: (1)administering to the subject at least one pharmaceutical agent that iscapable of inducing apoptosis and at least one plasmonics-active agent;and (2) applying an initiation energy from an initiation energy sourceto the subject, wherein the plasmonics-active agent enhances or modifiesthe applied initiation energy, such that the enhanced or modifiedinitiation energy activates the activatable pharmaceutical agent insitu, thus causing the at least one pharmaceutical agent to induceapoptosis in vivo.
 439. A method for generating an auto-vaccine effectin a subject in vivo, comprising: (1) administering to the subject atleast one pharmaceutical agent causing non-lytic cell death and at leastone plasmonics-active agent; and (2) applying an initiation energy froman initiation energy source to the subject, wherein theplasmonics-active agent enhances or modifies the applied initiationenergy, such that the enhanced or modified initiation energy activatesthe activatable pharmaceutical agent in situ, thus inducing, in vivo, anauto-vaccine effect in the subject that reacts with a target in thesubject.